Methods for treating a fibrin encapsulated tumor using a fibrin specific monoclonal antibody and compositions used therein

ABSTRACT

The subject invention relates a method for the production of monoclonal antibodies. The method utilizes an immunized germfree animal. The invention also provides methods for the use of such monoclonal antibodies, and polyclonal antibodies derived from an immunized germfree animal, for in vitro and in vivo clinical diagnostics and therapeutics. Also, the subject invention provides a fibrin-specific monoclonal antibody.

This is a division of application no. U.S. Ser. No. 086,423, filed Jul.2, 1993, now U.S. Pat. No. 5,453,359, which is a continuation-in-part ofapplication U.S. Ser. No. 081,914, filed Jun. 22, 1993, now abandoned,which is a continuation of application U.S. Ser. No. 835,800, filed Feb.14, 1992, now U.S. Pat. No. 5,223,410 which is a continuation ofapplication U.S. Ser. No. 364,053, filed Jun. 8, 1989, now U.S. Pat. No.5,120,834, which is a continuation-in-part of application U.S. Ser. No.206,259, filed Jun. 13, 1988, now abandoned.

1. FIELD OF THE INVENTION

The subject invention relates a method for the production of monoclonalantibodies. The method utilizes an immunized germfree animal. Theinvention also provides a method for the use of such monoclonalantibodies, and polyclonal antibodies derived from an immunized germfreeanimal, for in vitro and in vivo clinical diagnostics and therapeutics.Also, the subject invention provides a fibrin-specific monoclonalantibody.

2. BACKGROUND OF THE INVENTION

Kohler and Milstein are generally credited with having devised thetechniques that successfully resulted in the formation of the firstmonoclonal antibody-producing hybridomas (G. Kohler and C. Milstein,1975, Nature 256, 495-497; 1976, Eur. J., Immunol. 6, 511-519). Byfusing antibody-forming cells (spleen B-lymphocytes) with myeloma cells(malignant cells of bone marrow primary tumors) they created a hybridcell line, arising from a single fused cell hybrid (called a hybridomaor clone). The hybridoma had inherited certain characteristics of boththe lymphocytes and the myeloma cell lines. Like the lymphocytes, thehybridoma secreted a single type of immunoglobulin; moreover, like themyeloma cells, the hybridoma had the potential for indefinite celldivision. The combination of these two features offered distinctadvantages over conventional antisera.

Antisera derived from vaccinated animals are variable mixtures ofpolyclonal antibodies which never can be reproduced identically.Monoclonal antibodies are highly specific immunoglobulins of a singletype. The single type of immunoglobulins secreted by a hybridoma isspecific to one and only one antigenic determinant, or epitope, on theantigen, a complex molecule having a multiplicity of antigenicdeterminants. For instance, if the antigen is a protein, an antigenicdeterminant may be one of the many peptide sequences (generally 6-7amino acids in length; M. Z. Atassi, 1980, Molec. Cell. Biochem. 32,21-43) within the entire protein molecule. Hence, monoclonal antibodiesraised against a single antigen may be distinct from each otherdepending on the determinant that induced their formation; but for anygiven hybridoma, all of the antibodies it produces are identical.Furthermore, the hybridoma cell line is easily propagated in vitro or invivo, and yields monoclonal antibodies in extremely high concentration.

A monoclonal antibody can be utilized as a probe to detect its antigen.Thus, monoclonal antibodies have been used in in vitro diagnostics, forexample, radioimmunoassays and enzyme-linked immunoassays (ELISA), andin in vivo diagnostics, e.g. in vivo imaging with a radiolabeledmonoclonal antibody. Also, a monoclonal antibody can be utilized as avehicle for drug delivery to such antibodies' antigen.

However, before a monoclonal antibody can be utilized for such purpose,it is essential that the monoclonal antibody be capable of binding tothe antigen of interest; i.e., the target antigen. This procedure iscarried out by screening the hybridomas that are formed to determinewhich hybridomas, if any, produce a monoclonal antibody that is capableof binding to the target antigen. This screening procedure can be verytedious in that numerous, for example, perhaps several thousand,monoclonal antibodies may have to be screened before a hybridoma thatproduces an antibody that is capable of binding the target antigen isidentified. Accordingly, there is the need for a method for theproduction of monoclonal antibodies that increases the likelihood thatthe hybridoma will produce an antibody to the target antigen.

3. SUMMARY OF THE INVENTION

The subject invention provides a method for the production of monoclonalantibodies to an antigen comprising:

(a) immunizing a germfree animal with said antigen to permitantibody-producing cells to produce antibodies to said antigen,

(b) removing at least a portion of said antibody-producing cells fromsaid germfree animal,

(c) forming a hybridoma by fusing one of said antibody-producing cellswith an immortalizing cell wherein said hybridoma is capable ofproducing a monoclonal antibody to said antigen,

(d) propagating said hybridoma, and

(e) harvesting the monoclonal antibodies produced by said hybridoma.

The subject invention also provides methods for utilizing a monoclonalantibody or a polyclonal antibody derived from a germfree animal. Thesubject invention also provides a fibrin-specific monoclonal antibodyand methods for utilizing such a monoclonal antibody.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1(A) Determination of immunoreactivity of MH1 with DesAABB fibrinmonomer by affinity chromatography.

FIG. 1(B) Control experiment to confirm the binding capacity of theSepharose DesAABB fibrin monomer matrix using the fibrinogen specificantibody 45J.

FIG. 2 Immunoreactivity of non-crosslinked DesAABB fibrin polymers andcrosslinked DesAABB fibrin polymers in the in vitro assay for solubleDesAABB fibrin polymers using MH1.

FIG. 3 The formation of DesAA fibrin by batroxobin treatment of plasmafibrinogen.

FIG. 4 Immunoreactivity of soluble DesAA fibrin polymers in the in vitroassay system for soluble DesAABB fibrin polymers using MH1.

FIG. 5 Immunoreactivity of MH1 antibody with activated Factor XIII(Factor XIIIa)--treated fibrinogen.

FIG. 6 Measurement of soluble DesAABB fibrin polymer formation in vitrousing the soluble fibrin MH1 assay.

FIG. 7 Demonstration of the purification of soluble fibrin from plasmaby utilization of a Sepharose-MH1 column.

FIG. 8 Measurement of soluble DesAABB fibrin polymers in the blood ofpatients with chest pain and confirmed myocardial infarction.

4. DETAILED DESCRIPTION OF THE INVENTION 4.1. THE GERMFREE ANIMAL

The subject invention relates to the use of a germfree animal for theproduction of monoclonal antibodies. Germfree animals were firstdeveloped in the latter part of the 19th century and have been utilizedextensively since such time.

A germfree animal is a gnotobiote that is free from all demonstrableassociated forms of life, including bacteria, viruses, fungi, protozoa,and other saprophytic or parasitic forms. A gnotobiote is an animal orstrain derived by aseptic cesarean section or sterile hatching of eggsthat is reared and continuously maintained with germfree techniquesunder isolator conditions and in which the composition of any associatedfauna and flora, if present, is fully defined by accepted currentmethodology. (It should be noted that all mice carry a latentleukemogenic virus and, therefore, a mouse that would be germfree butfor such leukemogenic virus shall be considered a germfree animal forthe purpose of the subject invention.)

The essence of a germfree system is the provision of barriers againstthe entry of unwanted microbial invaders. In addition to the physicalbarriers of plastic, metal, rubber and glass which enclose the animals,the system requires the operational barriers of air filtration, food andwater sterilization, manipulation by gloves, which form an integral partof the barrier system. Also, the entry of supplies to the isolatorshould be performed under sterile conditions.

It is believed that any germfree animal ca n be utilized in the subjectinvention. The most common germfree animals are mouse, pig, rat, rabbit,guinea pig, goat, sheep, primate and poultry with a mouse beingpreferred, especially a Balb/C mouse.

4.2. PRODUCTION, CARE AND MAINTENANCE OF GERMFREE ANIMALS

There have been numerous publications concerning the production, careand maintenance of germfree animals. For example, Wostmann, B. S., Ed.,Gnotobiotes: Standards and Guidelines for the Breeding, Care andManagement of Laboratory Animals, National Research Council, NationalAcademy of Sciences, Washington, D.C. 1970; Coates, M. E., et al., TheGermfree Animal in Research, Academic Press, London, 1968; andPleasants, J. R., Gnotobiotics, in Handbook of Laboratory AnimalScience, Vol., 1, Melby, E. C., et al., Eds., CRC Press, Boca Raton,Fla., 117, (1974) the disclosure of which is incorporated herein byreference.

What follows is a summary derived from the article Wostmann, B. S. ed.,(1970) Gnotobiotes Standards and Guidelines for the Breeding, Care andManagement of Laboratory Animals, National Research Council, NationalAcademy of Sciences, Washington, D.C. describing the production, careand maintenance of germfree rats and mice. It should be noted that suchproduction, care and maintenance is similar for other animals.

ROOM ENVIRONMENT

The facilities, equipment, and husbandry procedures shall be designedand operated so as to afford maximum environmental control and optimalcomfort and welfare for the animals. The cages, feeders and waterersshall be so designed and fabricated as to afford maximum comfort for theanimals, to make the food and water readily available, and to makecleaning and sterilization practicable and efficient.

A desirable floor plan for extensive germfree work should consist of:

1. a work area for assembling and sterilizing the isolators,

2. an area for maintaining the isolators with animals, and

3. a laboratory area for the routine monitoring of the gnotobioticenvironment.

An office and diet-preparation area may be incorporated in the floorplan.

The room environment for maintaining gnotobiotic isolators should meetthe standards established for housing conventional laboratory rodents.The structure should be insect-proof, and the walls and floor should bemoisture-proof. Lighting should be uniform, with the same light-darkcycle throughout the year. Ventilation should rapidly remove any fumescaused by chemical sterilization, and the climate should be controlledas specified below.

Temperature.

The generally accepted animal room temperature of 21°-27° C. (70°-80°F.) may need to be adjusted downward to keep the isolator temperaturebetween 22° and 26° C. (72° and 78° F.).

Humidity.

The relative humidity (RH) should be kept at the human comfort level of40-60 percent. However, when room air is used to ventilate the isolator,40-50 percent RH is recommended.

Ventilation.

The room-air changes should be sufficient to remove rapidly any fumesgenerated during chemical sterilization. Ten to fifteen air changes perhour are recommended. Head masks with fresh-air ventilation should beavailable to protect personnel exposed to dangerous levels of chemicalfumes.

GERMFREE EQUIPMENT

(See Sacquiet, E. 1968, Equipment design and management: Generaltechnique of maintaining germ-free animals, p. 1-22 In M. E. Coates ed!.the germfree animal in research. Academic Press, London; Trexler, P. C.1968. Equipment design and management: Transport of germ-free animalsand current developments in equipment design, p. 23-35 In M. E. Coatesed!. The germfree animal in research. Academic Press, London)

Complete exclusion of environmental microbes requires an absolutebarrier. The successful operation of the isolator depends on themaintenance of that barrier at all times. There are two general types ofisolators available, metal and plastic. Some metal units are built towithstand internal steam pressure of 20 psi (1,406 g/cm²). (SeeReyniers, J. A. 1959. Design and operation of apparatus for rearinggerm-free animals. Ann. N.Y. Acad. Sci. 78:47; Miyakawa, M. 1959. TheMiyakawa remote-control germfree rearing unit. Ann. N.Y. Acad. Sci.78:37). Others are generally placed in a large autoclave for initialsterilization (See Gustafsson, B. E. 1959. Lightweight stainless steelsystems for rearing germfree animals. Ann. N.Y. Acad. Sci. 78:17.).

The flexible-film isolator (See Trexler, P. C., and L. I. Reynolds.1957. Flexible film apparatus for the rearing and use of germfreeanimals. Appl. Microbiol. 5:406) is now the most widely used unit. It isusually made of flexible laminated vinyl, must be chemically sterilized,and is readily adapted to specific needs. Another type, made from alarge tube of nylon, tied at each end, can be sterilized in anautoclave. (See Lev, M. 1962. An autoclavable plastic unit for rearinganimals under germfree conditions. J. Appl. Bacteriol 25:30). Plexiglassisolators and disposable flexible-film units also have been developed.Many of these are light enough to be stacked two or three high on arack, a feature that conserves floor space.

A special cylinder for sterilizing food and supplies is generally usedwith the heat-sensitive isolators. It should be designed with a largefiltration area to facilitate air removal in a high-vacuum autoclave(See Trexler, P. C. 1963. An isolator system for control ofcontamination. Lab. Anim. Care. 13:572). Alternatively, the cylinder maybe fitted with a drain tube vented to the atmosphere for removal of airand condensation during sterilization without the benefit of a vacuum.(See Jaworski, N. E., and C. E. Miller. 1963. Refinement of the cylindertechnique for supplying germfree isolators. Lab. Anim. Care. 13:591).

STERILIZATION

All equipment, food, bedding, water, and air used in the isolator mustbe absolutely sterile. The methods and conditions employed aredetermined by characteristics of the individual items.

Steam under pressure is the best-known method of sterilization. It isparticularly suitable for porous items that are heat-stable. Every areathat can conceivable harbor microbes must be brought into direct contactwith steam. Exposure time is related to the temperature used. It isrecommended that the least accessible portion of the load (the center ofthe packages) be exposed for a minimum period of 15 minutes at 121° C.(250° F.). Higher temperatures and shorter exposure periods may be usedafter careful testing to ensure sterility. Standard package size anddensity of diet, bedding, and other materials are of primary importanceto assure that the steam penetration time will be constant andpredictable.

Dry heat has been used for sterilization of the air supply for theisolator (See Miyakawa, M. 1959. The Miyakawa remote-control germfreerearing unit. Ann. N. Y. Acad. Sci. 78:37; Gustafsson, B. E. 1959.Lightweight stainless steel systems for rearing germfree animals. Ann.N.Y. Acad. Sci. 78:17).

Peracetic acid (CH₃ COOOH) is widely used on heat-sensitive, non-porousmaterials, especially the flexible-film units. This acid is used in a 2percent solution with a wetting agent (detergent) (See Trexler, P. C.,and L. I. Reynolds. 1957. Flexible film apparatus for the rearing anduse of germfree animals. Appl. Microbiol. 5:406). Other chemicals can beused for special situations, e.g., hypochlorites, iodophors, orquarternary ammonium compounds in the liquid trap to introduce newbornsobtained by hysterectomy, or HgC1₂ to introduce eggs under sterileconditions prior to hatching.

Ethylene oxide (ETO) may be used to sterilize nonwettable heat-sensitiveitems. Sterilization time is dependent on the temperature, humidity,pressure, and concentration of ETO. ETO may react chemically withbedding and dietary components to produce toxic or undesirablecompounds. Because of its flammability and toxic hazards, routine use ofETO for sterilization should be restricted to the commercially availablegas mixtures, which contain not more than 20 percent ETO.

Fiberglass filters are commonly used for sterilization of the airsupply. They should function as absolute filters.

Membrane filtration of liquids can be used to avoid exposure to heat,provided these membranes are absolute filters, for example, a filterthat excludes particles greater than 0.22 micrometers in diameter.

Irradiation by gamma rays or electron-beam sources may be used tosterilize diets or other special items. Dosages employed vary from 2.5to 6×10⁶ rads.

INTERNAL ENVIRONMENT

Temperature. The internal isolator temperature is a function of the roomenvironment and should be maintained between 22° and 26° C. (72°-78°F.).

Humidity.

The isolator is subject to condensation of moisture in cases ofoverloading, inadequate ventilation, or both. Air entering the isolatorshould be below 50 percent RH and preferably above 40 percent RH.

Air Supply.

The isolator should have 12 to 20 air changes per hour and a positivepressure of 3-5 in. (8-13 cm) of water. Air may be supplied from acentral source or from individual blowers for each unit. A turbine-typeair compressor is recommended for a central air supply system becausethe oil piston type tends to atomize oil into the air-supply lines.

An air diffusion isolator (See Trexler, P. C. 1968. Equipment design andmanagement: Transport of germ-free animals and current developments inequipment design, p. 23-35 In M. E. Coates ed!. The germfree animal inresearch. Academic Press, London) is not subject to loss of ventilationin the event of power failure. However, this type has the disadvantageof fewer air changes per hour and lacks the protective positive pressurethat could help prevent contamination should small breaks occur in thebarrier.

Emergency Safeguards.

Adequate provisions for the maintenance of air pressure within theisolator in the event of power failure or mechanical failure must beprovided with not more than a few minutes' interruption in the airsupply. Collapse of unsupported film isolators may eventually result insuffocation of the animals, but the more immediate danger is that theanimals may be able to reach and damage film or gloves. This may beprevented temporarily by plugging air conduits with rubber stoppers. Theoperation of individual isolator air supplies requires only an emergencypower supply. A central air system should have a second turbinecompressor for standby air supply.

Graphic recording of the temperature and pressure is recommended. Anaudiovisual alarm system should be incorporated in a central air systemto be actuated by a drop in line pressure in the event of either loss ofpower or mechanical failure. Similar alarm systems should indicateundesirable fluctuations in the temperature of the air supply. Forindividual isolator air systems, continuous graphic monitoring of theroom environment is recommended.

CAGING AND INTERIOR EQUIPMENT

Equipment.

A basic list of equipment for an isolator may include cages with securelids, water bottles and food hoppers, protective cloth gloves for therubber gloves, an extra door gasket or cap closing ring, longrubber-tipped forceps, hemostats, scissors, a towel, gauze sponges, atwo-quart can for holding instruments, a covered four-quart diet can,spoon, culture tubes, paper bags, and moisture-resistant bags for dirtybedding.

Cages should be fabricated of a smooth corrosion-resistant material.They should be impervious to liquids and easily sterilized. Materialsconsidered acceptable include plastics, stainless steel, and glass.Galvanized metal becomes corroded and is not recommended becausetrace-metal contamination may influence experimental results.

Cage dimensions are usually limited by the size of the entry port. Theminimum area for a female mouse and litter is 50 in.² (970 cm²). In manycircumstances more space per animal may be needed.

Table 1 lists the recommended floor space per animal for mice and ratsaccording to weight groupings.

                  TABLE 1    ______________________________________    Amount of Floor Space Recommended per Animal    for Caged Mice and Rat                                     Maximum    Category             Space per Animal                                     Population    Number    Weight (g) in..sup.2 (cm.sup.2)                                     Per Cage    ______________________________________    MICE    1         up to 10   6 (40)      40    2         10-15      8 (50)      30    3         15-25      12 (75)     20    4         over 25    15 (95)     16    RATS    1         up to 50   15 (95)     50    2         50-100     17 (110)    50    3         100-150    19 (125)    40    4         150-200    23 (150)    40    5         200-300    29 (185)    30    6         over 300   40 (260)    25    ______________________________________

MISCELLANEOUS RECOMMENDATIONS

Freon tests for minute leaks are recommended to ensure the integrity ofthe barrier system.

Each unit should be equipped with its own operation log to maintain achronological record of every procedure involving the unit from the timeit is assembled and sterilized. Such records are conveniently kept inmetal hospital-chart holders identified by the isolator number. Theyshould also contain notes for routine maintenance, e.g., glovereplacement. Breeding-performance records may be kept in the same chartholder.

Due to the limited space available inside the isolators, paper andfolding containers are recommended for diets and bedding, and for thetransport of animals between isolators linked by a sterile passage.

No ether should be used inside an isolator because it may explode whenstatic sparks occur. Fluothane (bromochlorotrifluoroethane) isrecommended as a volatile, nonflammable anesthetic.

DIETS, BEDDING AND WATER

GENERAL RECOMMENDATIONS

The complete formula for commercially produced diets should be provided,listing all the ingredients and their concentrations, includingpreservatives, antioxidants, and other additions. The date of productionshould be clearly indicated. The manufacturer should guarantee that thediet is:

1. Within the normal acceptable limits of naturally occurring hormoneactivity.

2. Free of additives containing drugs, hormones, antibiotics, or anyother substance that may create abnormal physiological conditions orinterfere with investigative procedures.

3. Free of salmonella on the basis of statistically selected samples.

4. Free of rodent and vermin contamination.

5. Free of all unrendered meat scraps or fish meal that may containpathogens.

FORTIFICATION OF DIETS

Diets of germfree animals must contain more than normal requirements ofcertain nutrients to compensate for the heat-sterilization loss ofvitamins (especially certain B vitamins and vitamins A and D) and of thenutritive value of protein (reduction in available lysine, methionine,arginine and tryptophan). They must also provide required nutrients,which in conventional animals would be available through microbialsynthesis in the gastrointestinal tract (See Reddy, B. S., B. S.Wostmann, and J. R. Pleasants. 1968 Nutritionally adequate diets forgerm-free animals, p. 87-111 In M. E. Coates ed!. the germ-free animalin-research. Academic Press, London). An example of such a diet isL-485, an inexpensive diet that has been extensively tested (SeeKellogg, T. F., and B. S. Wostmann. 1969. Rat and mouse stock dietL-485. Lab. Anim. Care.) and can be commercially produced (see Table 2).Supplementation with specific amino acids rather than increased totalprotein content should be considered as a means to compensate for lossin protein quality. Increasing the total protein content of the dietwill result in a greater consumption and excretion of water, causinghumid conditions and thereby limiting the number of animals that can behoused in an isolator of a given size.

                  TABLE 2    ______________________________________    Composition of Diet L-485 for Rats and Mice    Ingredient              Amount per kg    ______________________________________    DIET    Ground yellow corn (maize)                            590 g    Soybean oil meal (crude protein 50 percent)                            300    Alfalfa meal (dehydrated; 7 percent protein)                            35    Corn oil (once refined) 30    NaCl                    10    CaHPO.sub.4 2H.sub.2 O  10    CaCO.sub.3              5    Lysine (feed grade)     5    Methionine (feed grade) 5    B.H.T. (butylated hydroxytoluene)                            0.125    Trace mineral mix       0.25    VITAMIN MIX    A                       26,000 IU    D.sub.3                 1,000 IU    E (a tocopherol acetate)                            225 mg    K.sub.3 (menadione)     90    Riboflavin              30    Pantothenic acid        285    Niacin                  65    Choline chloride        2,000    B.sub.12 (0.1 percent trituration in mannitol)                            2    Thiamine HCl            65    Pyridoxine HCl          20    Folic acid              10    Para-aminobenzoic acid  50    TRACE MINERAL MIX (commercial    MN as manganous oxide   65 mg    Fe as ferrous carbonate 20    Cu as copper oxides     2    Zn as zinc oxide        15    I as calcium iodate     1.5    Co as cobalt carbonate  0.6    ______________________________________

Steam sterilization (See Reddy, B. S., B. S. Wostmann, and J. R.Pleasants. 1968 Nutritionally adequate diets for germ-free animals, p.87-111 In M. E. Coates ed!. the germ-free animal in research. AcademicPress, London)

Actual procedures will depend on the equipment available. Three factorsare of general importance:

1. A pre-sterilization vacuum, whenever possible, of at least 20 in. Hgwill assist steam penetration of the diet in a clave or cylinder ventedto the atmosphere. A vacuum of 28 in. Hg or more is recommended when thesupply cylinder is not vented to the atmosphere.

2. Use of the shortest sterilization phase that will ensure totalsterility, with an added safety margin dictated by equipment and skill.Temperatures measured at the inner core of the diet should reach atleast 121° C. (250° F.). At that temperature the actual sterilizationphase should last a minimum of 15 minutes. With higher sterilizationtemperatures, sterilization times will be relatively shorter.

3. A post-sterilization vacuum will speed the reduction of temperatureof the diet. This will avoid unnecessary heat destruction of nutrients.However, the design and performance of the apparatus must be adequate toavoid leaks during this stage of the operation.

In steam sterilization of diets, the goal is to avoid both incompletesterilization and unnecessary nutritional damage caused by excessivelyprolonged heating. Although some nutrient loss is unavoidable, quiteacceptable results may be obtained by manipulation of:

(a) Technical procedures such as temperature, time pre-sterilization andpost-sterilization vacuum, and pellet size.

(b) The water content of the diet. An increase in water content leads tobetter recovery of B vitamins after sterilization (See Zimmerman, D. R.,and B. S. Wostmann. 1963. vitamin stability in diets sterilized forgermfree animals. J. Nutr. 79:318). For solid diets, a water content upto 25% percent, or as high as proves to be compatible with the storagequality of the diet, is recommended. A change in water content of thediet should be followed by a new test of the rate at which the dietreaches sterilizing temperature.

RADIATION STERILIZATION

(See Reddy, B. S., B. S. Wostmann, and J. R. Pleasants. 1968.Nutritionally adequate diets for germ-free animals, p. 87-111 In M. E.Coates ed!. The germ-free animal in research. Academic Press, London;Ley, F. J., J. Bleby, M. E. Coates, and J. S. Paterson. 1969.Sterilization of laboratory animal diets using gamma radiation. Lab.Anim. 3:221)

Techniques and dosimetry will depend on equipment and type of radiation.Although, in general, radiation sterilization is considered to result inless destruction of nutrients, it is at present recommended that dietsbe sterilized with steam.

TEST FOR STERILITY

To monitor sterility achieved with any specific sterilization procedure,the use of Bacillus stearothermophilus spore strips is recommended. Thestrips should be embedded in the core of the diet. Also, the isolatorand its animals should be periodically microbiologically monitored. Suchmonitoring is necessary to test for accidental contaminations resultingfrom breaks in the isolator barriers or from inadequate sterilization ofthe isolator or its contents. This can be accomplished as described inWostmann, B. S., Ed., Gnotobiotes: Standards and Guidelines for theBreeding Care and Management of Laboratory Animals, National ResearchCouncil, National Academy of Sciences, Washington, D.C., 1970, pp.28-39.

ESTIMATION OF NUTRIENT LOSS DURING STERILIZATION

As a useful check on the loss of vital nutrients, determination ofacid-extractable thiamine as an indicator of the recovery of thiamineadded to the diet is recommended (See Wostmann, B. S. and P. L. Knight.1960. The effect of methyl alcohol on the conversion of thiamine tothiochrome. Experientia 16:500). A recovery of less than 25 percentindicates severe impairment of general nutritional quality of the diet.With adequate equipment and care, recoveries of 50 percent or moreshould be achieved.

STORAGE OF SOLID DIET

Because of the generally high cost of germfree experimentation, extracare should be taken never to use diet that has decreased significantlyin nutritional value. It is recommended that (a) nonsterilized dietalways be stored under refrigeration, and never for longer than onemonth, and that (b) storage time of sterilized diet inside the isolatorshould be one week or less and must never exceed ten days.

BEDDING

Bedding should be changed at least once a week. It is recommended thatbedding material be easy to sterilize and not readily eaten by theanimals. It should not yield toxic compounds as a result of thesterilization procedure. Dustfree white pine chips (sawdust) andshavings are recommended. Basswood and poplar shavings or crushed corncobs are acceptable. Diatomaceous products, cedar, resinous woods, andhardwoods are not recommended. Ethylene oxide sterilization should notbe used until the question of possible formation of harmful compoundshas been clarified.

WATER

Drinking water must be sterilized. It may be autoclaved in square packflasks, Mason jars, or tanks attached to the unit. A small air spaceshould be left inside each container.

PRINCIPLES OF CESAREAN DERIVATION OF GNOTOBIOTES

The success of any cesarean operation is keyed in part to having thepregnancy advance to full term. This is particularly true of animalswith short gestation periods, where the fetus may gain 20 percent of itsweight in the final 24 hours before parturition. Timed matings arereasonably successful, but with animals yielding large litters (rats andmice) it may be helpful to wait for the female to deliver the firstoffspring before proceeding with the operation. In guinea pigs, the mostsatisfactory method is to select females for surgery by measuring thespread of the pubic bones (See Philips, B. P., P. A. Wolfe, and H. A.Gordon. 1959. Studies on rearing the guinea pig germfree. Ann. N.Y.Acad. Sci. 78:183).

The cesarean-derived young must be delivered into a germfree environmentbefore they take their first breath of air. They may be taken directlyfrom the mother by hysterotomy, through an incised sterile barriermembrane into a sterile isolator, or by hysterectomy, through agermicidal trap into a sterile isolator. The usual surgical preparationof the female prior to the cesarean operation includes removal ofabdominal hair and cleaning and disinfection of the operative site.Anesthesia is accomplished preferentially by dislocation of the cervicalvertebrae in rats and mice, although an abdominal midline localanesthetic or general anesthesia may also be used without incurringserious levels of fetal depression and mortality. With guinea pigs,surgery is generally performed after prior sedation and under localanesthesia.

Delivery of the young by hysterotomy through a barrier membrane requiresspecial isolator equipment. The Reyniers stainless-steel surgical unit(See Reyniers, J. A. 1965. Germfree life methodology (gnotobiotics) andexperimental nutrition. p. 458-466 In Proc. 3rd Internat. Congr.Biochem., Bruxelles) has a built-in horizontal metal divider thatseparates the upper and lower compartments of the unit. The dividercontains a circular port covered with a mylar plastic film to maintainthe integrity of the upper compartment. The female, prepared forsurgery, is placed in the lower compartment with the abdomen pressedagainst the mylar. All surgical instruments are in the uppercompartment, and the surgery is performed in this sterile area. Anincision is made through the plastic and skin with an electrocautery orscalpel. The self-sterilizing electrocautery blade is preferred for skinincision. The edges of the skin and mylar are clamped together andreflected. A sterile drape is placed over the abdomen to cover the cutedges of the skin, and warm disinfectant (benzalkonium chloride 1:1,000)is applied to the exposed fascia before opening the abdominal cavity.Extreme caution must be exercised to avoid cutting into the bowel. Theinsertion of a pair of forceps or hemostats between the peritoneal walland the viscera may be helpful. The uterus is then opened and the youngremoved. The fetal membranes are removed, and the umbilical cord isclamped and cut. The young are gently dried and massaged to stimulaterespiration. They are then transferred to a rearing unit to befoster-nursed or hand-fed. Another sheet of mylar may be secured overthe surgical port and the procedure repeated with as many as 5 or 6females without serious risk of contamination. Cesarean delivery mayalso be accomplished using a plastic isolator or glove bag as a surgicalunit. The exterior surface of the isolator floor is presterilized andbrought into contact with the animal's abdomen, thus serving the samepurpose as the mylar sheet described above. Following the operation theslit in the plastic barrier can be closed with sterile tape and thesurgical procedure repeated on additional gravid females.

Delivery of the young by hysterotomy is more common when plasticisolators are used (See Foster, H. 1959. A procedure for obtainingnucleus stock for a pathogen-free animal colony. Lab. Anim. Care 9:135).The uterus is aseptically exposed and clamped just anterior to thecervix. The excised uterus is transferred into the germfree unit througha liquid germicidal trap. Once inside the isolator, the young aredelivered as rapidly as possible to prevent aspiration of fetal fluids.Normally they are dried and breathing well before the umbilical cord isclamped and cut. The infants are then given to the foster mother or handreared.

Hysterotomy may be used successfully for mice, rats, and swine. Inguinea pigs, however, hysterotomy is preferred, since a high mortalityoccurs if as much as two minutes elapses between the severance from thematernal blood supply and delivery inside the isolator.

BREEDING SYSTEMS IN GNOTOBIOTIC COLONIES INBRED STRAINS

The usual brother X sister mating system employed in conventionalbreeding colonies can also be used in gnotobiotic colonies.

NONINBRED STOCKS

True random breeding includes some matings of siblings and of firstcousins. Although such matings are normally avoided in noninbredbreeding colonies, the resulting mating system does not decrease therate of inbreeding to the maximum extent possible. Any system of minimalinbreeding can be used. (See Falconer, D. C. 1967. Genetic aspects ofbreeding methods. p. 72-96 In The UFAQ handbook on the care andmanagement of laboratory animals, 3rd ed. E and S Livingstone, LTd.,London; National Research Council, Institute for Laboratory AnimalResources. 1969. A guide to genetic standards for laboratory animals.National Academy of Sciences, Washinton, D.C.).

Comparable Conventional and Gnotobiotic Colonies.

If it is desired to maintain both conventional and gnotobiotic coloniesfor comparative purposes, their similar genetic constitutions may bemaintained by introducing cesarean-derived litters from the effectivebreeding population of the conventional colony into the gnotobioticcolony. This appears to be the method of choice for those producers whoplace most emphasis on the production of nongnotobiotic rats and micebut has the disadvantage of preventing the establishment of amicrobiological pedigree that would simplify microbiological monitoring.Ideally, this could be done by using litters from specific matings asbreeding stock for the conventional colony and using the next litterfrom each of these matings as breeding stock for the gnotiobioticcolony. If this procedure is followed every second or third generation,the genetic constitutions of both colonies should remain very similar,provided, of course, that the same mating system is used in each colony.

Alternatively, litters from the effective breeding population of thegnotobiotic colony may be used to establish or replenish theconventional colony. The genetic consequences will be identical,provided the same procedures are followed.

RECORD-KEEPING

(See Wolff, G. L. 1967. Practical mating systems and record-keeping in abreeding colony. p. 97-113 In the UFAW handbook on the care andmanagement of laboratory animals, 3rd ed. E. and S Livingstone, Ltd.,London).

Proper records should be kept for the animals and for the maintenance ofthe isolator. The animals' records should determine the efficiency ofthe operation and the biological performance of the animals. Theisolator records should maintain a chronology of events related to theisolator to assist in locating a breach of the barrier if contaminationoccurs.

4.3. THE ANTIGEN-FREE ANIMAL

In a preferred embodiment of the subject invention, it is preferred thatthe germfree animal be bred on a chemically defined (CD), low molecularweight, water-soluble, ultrafiltered diet. It is believed that such adiet permits one to obtain complete control of nutrient and antigenintake by the animal. Such a diet is generally made up entirely ofingredients that are capable of chemical definition, e.g., amino acids,simple sugars, lipids, vitamins and minerals. For the purpose of thesubject invention a chemically defined diet comprises amino acids,simple sugars, lipids, vitamins and minerals and no other componenthaving a molecular weight greater than about 10,000 daltons. Thus, allof the components of a CD diet are of low molecular weight and arenaturally circulating nutrients in animals and, therefore, it isbelieved that such components will not stimulate an immune response. Therecent literature refers to a germfree animal that has been bred on a CDdiet as an "antigen-free animal".

Also, it is preferred to utilize a filter paper bedding, otherwise thegermfree animal may eat the bedding, which results in an immuneresponse. It is believed that the eating of a filter paper bedding doesnot result in an immune response.

The particular CD diet for a given species would use such components inproportions and quantities so as to fulfill known nutritionalrequirements for such species. The composition and preparation of apreferred CD diet for germfree mice is as follows:

    ______________________________________    Composition and preparation of    chemically defined diet L489E14Se                             Amount                             (grams/100                             grams of    Ingredient               Ingredient)    ______________________________________    To 192 ml Milli-Q water at 70° C. the following are    added:    Leucine                  1.9    Phenylalanine            0.74    Isoleucine               1.08    Methionine               1.06    Tryptophan               0.37    Valine                   1.23    Asparagine               0.91    Arginine HCl             0.81    Threonine                0.74    Lysine HCl               1.77    Histidine HCl            0.74    The solution is cooled to 45° C. and the following    added:    Glycine                  0.59    Proline                  1.48    Serine                   1.33    Alanine                  0.59    Sodium glutamate         3.40    L-tyrosine ethyl ester HCl                             0.62    Ferrous gluconate        0.05    Salts 35D.sup.1          0.105    Sodium Selenite.sup.2    0.074    Solution cooled to 5° C. and the following added:    Calcium glycerophosphate 5.22    Magnesium glycerophosphate                             1.43    Calcium chloride 2H.sub.2 O                             0.185    Sodium chloride - Potassium Iodide    (KI) mix (containing    680 mg KI)               0.086    Vitamin B mix 111E5.sup.3                             0.09    Vitamin B12.sup.4        1.20 mg    Choline chloride         0.31    Potassium acetate        1.85    To 108 ml Milli-Q water at 70° C.    D-Dextrose, anhydrous was added                             71.28    Solutions cooled to 5° C. and combined both for    ultrafiltration.    ______________________________________     .sup.1 Composition of salts 35D mixture given in table 3     .sup.2 Added in addition to sodium selenate in Salts 35D     .sup.3 Composition of vitamin B mix111E5 as stated in Table 4     .sup.4 Added in addition to vitamin B12 in vitamin B mix 111E5 (see     EXAMPLE hereinbelow)

Such composition is fed to mice or rats ad libitum.

    ______________________________________    Composition of lipid supplement LADEK 69E6                         Amount per daily adult    Ingredient           dose* (0.385 ml)    ______________________________________    Purified soy triglycerides.sup.1                         0.33 g    Retinyl palmitate    6.45 mg (11.7 I.U.)    Cholecalciferol      0.0288 mg (1.15 I.U.)    2ambo-alpha-tocopherol                         3.3 mg    2ambo-alpha-tocopherol acetate                         6.6 mg    Phylloquinone        72 mg    ______________________________________     *Lacating mice receive twice the normal adult dose.     .sup.1 Consisting of:     12% palmitate     1.5% stearate     24% oleate     55% linoleate     8% linolenate

For a detailed description of CD diets see Pleasants, J. R., et al., J.Nutr., 116, 1949-1964 (1986), Pleasants, J., et al., Germfree Research:Microflora Control and Its Application to the Biomedical Sciences, B. S.Wostmann, Ed., p. 87, Liss, New York (1985); Wostmann, B. S., et al., J.Nutr., 112, 552 (1982); and Pleasants, J. R., et al., J. Nutr., 100, 498(1970), the disclosures of which are incorporated herein by reference.

4.4 PRODUCTION OF MONOCLONAL ANTIBODY

The germfree animal is then utilized for the production of monoclonalantibodies. The germfree system can be utilized to produce a monoclonalantibody to any antigen that the animal in a nongermfree state couldproduce. An examplary list of antigens appears in U.S. Pat. No.3,935,074. However, it is believed that the germfree animal provides amuch enhanced immune response to the antigen. Thus, one can increase thelikelihood of locating a B-lymphocyte that produces an antibody that iscapable of binding to a specific epitope of the antigen. This is a majoradvantage of the subject invention. In addition, it is believed that thegermfree system is particularly useful for generating a highly specificantibody for those antigens with numerous epitopes.

The germfree animal can be immunized by standard techniques. However, itis preferred that the germfree animal be immunized at least three timeswith at least about three weeks between each immunization, followed by aprefusion booster. It is believed that this increased level ofimmunization may be necessary because it has been observed that aftertwo immunizations, which is customary, there are still mostly IgMsecreting B-lymphocytes rather than the preferred IgG secretingB-lymphocytes.

4.5. SOMATIC CELLS

Somatic cells of the germfree animal having the potential for producingantibody and, in particular B lymphocytes, are suitable for fusion witha B-cell myeloma line. Those antibody-producing cells that are in thedividing plasmablast stage fuse preferentially. Somatic cells can bederived from the lymph nodes, spleens and peripheral blood of primedgermfree animals, and the lymphatic cells of choice depend to a largeextent on their empirical usefulness in the particular fusion system.However, somatic cells derived from the spleen are generally preferred.Once primed or hyperimmunized, germfree animals can be used as a sourceof antibody-producing lymphocytes. Mouse lymphocytes give a higherpercentage of stable fusions with the mouse myeloma lines describedhereinbelow. However, the use of antibody-producing cells from othergermfree animals is also possible. The choice of a particular germfreeanimal depends on the choice of antigen, for it is essential that thegermfree animal have a B-lymphocyte in its repertoire of B-lymphocytesthat can produce an antibody to such antigen.

4.6. IMMORTALIZING CELLS

Specialized myeloma cell lines have been developed from lymphocytetumors for use in hybridoma-producing fusion procedures (G. Kohler andC. Milstein, 1976, Eur. J. Immunol. 6:511-519; M. Schulman et al., 1978,Nature 276:269-270). The cell lines have been developed for at leastthree reasons. The first reason is to facilitate the selection of fusedmyeloma cells. Usually, this is accomplished by using myelomas withenzyme deficiencies that render them incapable of growing in certainselective media that support the growth of hybridomas. The second reasonarises from the inherent ability of lymphocyte tumor cells to producetheir own antibodies. The purpose of using monoclonal techniques is toobtain immortal fused hybrid cell lines that produce the desired singlespecific antibody genetically directed by the somatic cell component ofthe hybridoma. To eliminate the production of tumor cell antibodies bythe hybridomas, myeloma cell lines incapable of producing light or heavyimmunoglobulin chains or those deficient in antibody secretionmechanisms are used. A third reason for selection of these cell lines istheir suitability and efficiency for fusion.

Several myeloma cell lines can be used for the production of fused cellhybrids, including NS-1, X63-Ag8, NIS-Ag4/1, MPC11-45.6TG1.7,X63-Ag8.653, Sp2/0-Agf14, FO, and S194/5XXO.Bu.1., all derived frommice, and 210-.RCY3.Ag1.2.3 derived from rats. (G. J. Hammerling, U.Hammerling and J. F. Kearnly, eds., 1981, Monoclonal antibodies andhybridomas In J. L. Turk, eds. Research Monographs in Immunology, Vol.3, Elsevier/North Holland Biomedical Press, New York).

4.7. FUSION

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and immortalizing cells generally comprise mixing somaticcells with immortalizing cells in a proportion which can vary from about20:1 to about 1:1 in the presence of an agent or agents (chemical, viralor electrical) that promote the fusion of cell membranes. It is oftenpreferred that the same species of animal serve as the source of thesomatic and immortalizing cells used in the fusion procedure. Fusionmethods have been described by Kohler and Milstein (1975, Nature256:495-497; 1976, Eur. J. Immunol. 6:511-519), by Gefter et al. (1977,Somatic Cell Genet. 3:231-236) and by Kozbor et al., 1983, ImmunologyToday, 4, 72. The fusion-promoting agents used by those investigatorswere Sendai virus and polyethylene glycol (PEG), respectively.

One can also utilize the recently developed EBV-transformation technique(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96).

4.8. ISOLATION OF CLONES AND ANTIBODY DETECTION

Fusion procedures usually produce viable hybrids at very low frequency,about 1×10⁻⁶ to 1×10⁻⁸. Because of the low frequency of obtaining viablehybrids, it is essential to have a means to select fused cell hybridsfrom the remaining unfused cells, particularly the unfused myelomacells. A means of detecting the desired antibody-producing hybridomasamong the other resulting fused cell hybrids is also necessary.

Generally, the fused cells are cultured in selective media, for instanceHAT medium, which contains hypoxanthine, aminopterin and thymidine. HATmedium permits the proliferation of hybrid cells and prevents growth ofunfused myeloma cells which normally would continue to divideindefinitely. Aminopterin blocks de novo purine and pyrimidine synthesisby inhibiting the production of tetrahydrofolate. The addition ofthymidine bypasses the block in pyrimidine synthesis, while hypoxanthineis included in the media so that inhibited cells can synthesize purineusing the nucleotide salvage pathway. The myeloma cells employed aremutants lacking hypoxanthine phosphoribosyl transferase (HPRT) and thuscannot utilize the salvage pathway. In the surviving hybrid, the Blymphocyte supplies genetic information for production of this enzyme.Since B lymphocytes themselves have a limited life span in culture(approximately two weeks), the only cells which can proliferate in HATmedia are hybrids formed from myeloma and spleen cells.

To facilitate screening of antibody secreted by the hybrids and toprevent individual hybrids from overgrowing others, the mixture of fusedmyeloma and B-lymphocytes is diluted in HAT medium and cultured inmultiple wells of microtiter plates. In two to three weeks, when hybridclones become visible microscopically, the supernatant fluid of theindividual wells containing hybrid clones is assayed for specificantibody production.

The assay must be sensitive, simple and rapid. Assay techniques includeradioimmunoassays, enzyme immunoassays, cytotoxicity assays, and plaqueassays.

4.9. CELL PROPAGATION AND ANTIBODY PRODUCTION

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line can bepropagated in either of two standard ways. A sample of the hybridoma canbe injected into a histocompatible animal of the type that was used toprovide the somatic and myeloma cells for the original fusion. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can be tapped to providemonoclonal antibodies in high concentration. Alternatively, theindividual cell lines can be propagated in vitro in laboratory culturevessels. The culture medium, containing high concentrations of a singlespecific monoclonal antibody, can be harvested by decantation,filtration or centrifugation.

4.10. USE OF THE MONOCLONAL ANTIBODY

The monoclonal antibodies made by the method of the subject inventioncan be utilized in any technique known or to be developed in the futurethat utilizes a monoclonal antibody.

A major use of monoclonal antibodies is in an immunoassay, which is themeasurement of the antigen-antibody interaction. Such assays aregenerally heterogeneous or homogeneous. In a homogeneous immunoassay theimmunological reaction usually involves the specific antibody, a labeledanalyte, and the sample of interest. The signal arising from the labelis modified, directly or indirectly, upon the binding of the antibody tothe labeled analyte. Both the immunological reaction and detection ofthe extent thereof are carried out in a homogeneous solution.Immunochemical labels which may be employed include free radicals,fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth. Themajor advantage of a homogeneous immunoassay is that the specificantibody need not be separated from the labeled analyte.

In a heterogeneous immunoassay, the reagents are usually the specimen,the specific antibody, and means for producing a detectable signal. Thespecimen is generally placed on a support, such as a plate or a slide,and contacted with the antibody in a liquid phase. The support is thenseparated from the liquid phase and either the support phase or theliquid phase is examined for a detectable signal employing means forproducing such signal. The signal is related to the presence of theanalyte in the specimen. Means for producing a detectable signal includethe use of radioactive labels, fluorescers, enzymes, and so forth.Exemplary of heterogeneous immunoassays are the radioimmunossay,immunofluoroescence methods, enzyme-linked immunoassays, and the like.

For a more detailed discussion of the above immunoassay techniques, see"Enzyme-Immunoassay," by Edward T. Maggio, CRC Press, Inc., Boca Raton,Fla., 1980. See also, for example, U.S. Pat. Nos. 3,690,834; 3,791,932;3,817,837; 3,850,578; 3,853,987; 3,867,517; 3,901,654; 3,935,074;3,984,533; 3,996,345; and 4,098,876, which listing is not intended to beexhaustive.

Another major use of monoclonal antibodies are in-vivo imaging andtherapeutics. The monoclonal antibodies can be labelled with radioactivecompounds, for instance, radioactive iodine, and administered to apatient intravenously. The antibody can also be labelled with a magneticprobe. NMR can then be utilized to pinpoint the antigen. Afterlocalization of the antibodies at the antigen, the antigen can bedetected by emission tomographical and radionuclear scanning techniques,thereby pinpointing the location of the antigen.

By way of illustration, the purified monoclonal antibody is suspended inan appropriate carrier, e.g., saline, with or without human albumin, atan appropriate dosage and is administered intravenously, e.g., bycontinuous intravenous infusion over several hours, as in Miller et al.In Hybridomas in Cancer Diagnosis and Therapy (1982), incorporatedherein by reference.

The monoclonal antibodies of subject invention can be usedtherapeutically. Antibodies with the proper biological properties areuseful directly as therapeutic agents. Alternatively, the antibodies canbe bound to a toxin to form an immunotoxin or to a radioactive materialor drug to form a radiopharmaceutical or pharmaceutical. Methods forproducing immunotoxins and radiopharmaceuticals of antibodies arewell-known (see, for example, Cancer Treatment Reports (1984)68:317-328).

It also is believed that polyclonal antibodies derived from a germfreeanimal also can be utilized in immunoassays and provide an improvedresult as compared to polyclonal antibodies derived from a conventionalanimal. Polyclonal antibodies derived from a germfree animal can be madeby utilizing a germfree animal, as described hereinabove, andimmunization techniques, as described hereinabove, followed byseparating the polyclonal antibodies from the animal by conventionaltechniques, e.g. by separating the serum from the animal.

A FIBRIN-SPECIFIC MONOCLONAL ANTIBODY 5.1. BACKGROUND

The hemostatic mechanism is a complex physiological response mechanisminvolved in repairing damage to a ruptured blood vessel. Hemostasis isachieved through the co-operative interactions among the wall of thedamaged blood vessel, the platelets and the coagulative system. The roleof the coagulation system is to provide an extensive fibrin network tostabilize and anchor the platelet plug which has been assembled on thesubendothelial structure of the damaged vessel. The formation of theinsoluble fibrin matrix from circulating fibrinogen is the result of acomplex sequence of reactions culminating in the explosive production ofthrombin at the required site. Coagulation is an amplification processinvolving a chain of enzymatic reactions in which proenzymes (clottingfactors) are activated sequentially to active enzymes. There are anumber of physiological mechanisms controlling the fibrin polymerizationprocess involved in thrombus formation. These include the thrombininhibitor antithrombin III (ATIII), protein C, prostacyclin and variouscomponents of the fibrinolytic system such as tissue plasminogenactivator (t-PA) and its fast acting inhibitor (PAI).

The homeostasis hypothesis proposed by Astrup in 1956 Astrup, T., Blood11, 781-806 (1956) states than an equilibrium exists between fibrinformation (coagulation) and fibrin dissolution (fibrinolysis). In thenormal or healthy state these functions are evenly balanced. However,when the hemostatic process is impaired, coagulation and fibrinolysisare pathologically expressed as thrombosis and hemorrhage, respectively.The clinical manifestations of pathological thrombosis or thromboticdisease are extremely diverse and include disseminated intravascularcoagulation (DIC), deep vein thrombosis (DVT), arterial and venousthrombosis. Thromboembolism and thrombotic complications of othervascular disease (eg. atherosclerosis) can result in occlusion of majorarteries leading to organ ischemia and the attendant life-threateningconditions such as cerebrovascular accident (stroke), myocardialinfarction, etc.

The fibrinolytic process involves the conversion of an inactive zymogen,plasminogen, to the proteolytic enzyme, plasmin, through the action ofagents known as plasminogen activators. The molecular mechanism ofphysiological fibrinolysis is not fully understood, but it is known thatduring fibrin formation plasminogen binds to fibrin where it can beactivated by plasminogen activators, e.g. t-PA. In this manner plasmingeneration proceeds within the thrombus where it is protected frominactivation by the main physiological inhibitor of plasmin, alpha₂-antiplasmin.

Upon exposure to plasmin, fibrinogen and fibrin are broken down to theirdegradation products. Fibrinogen breaks down into fragments X and Y, andupon further exposure to plasmin, fragments D and E. Fibrin breaks downto fragments X, Y, D and E from non-crosslinked fibrin and crosslinkedD-dimer, D-D/E complex, Y dimer, Y-D-dimer and X oligomer fromcrosslinked fibrin.

Assays for markers of thrombotic disorders have been conducted untilquite recently using polyclonal antibodies in both radioimmunoassays andlatex agglutination type assays. These assays have been demonstrated tobe extremely unreliable by Gaffney (Gaffney, P. J., Ann. N.Y. Acad.Sci., 408, 407-423 (1983). More specific and sensitive immunoassays(such as ELISAs) using monoclonal antibodies are becoming commonpractice in clinical laboratories. The limiting factor in thesediagnostic assays is the specificity and affinity of the particularmonoclonal antibody employed. The generation of highly specificantibodies to any of the potential indicators of impaired hemostasis ishampered by both low levels of indicators and the antigenic relatednessof the particular marker with its precursor, which is normally presentat much higher levels in plasma. Examples are the formation of complexesbetween enzymes and their inhibitors eg. thrombin-antithrombin III,plasmin-alpha₂ -antiplasmin, t-PA-PAI-1. The number of new antigenicsites generated by such complex formation is extremely small and makesthe production of immunological probes (such as monoclonal antibodies)difficult.

Likewise, the major problem associated with the acquisition of amonoclonal antibody to fibrin has been the structural and conformationalsimilarities between fibrin and its physiological precursor fibrinogen.It has been estimated that the conservation of covalent structure whenfibrinogen is converted to fibrin is greater that 98% (Plow, E. F., etal., Semin. Thromb. Haemostas, 8, 36 (1982) and, therefore, only a smallpercentage of the epitopes on the fibrin molecule are in factneoantigens (and unique to fibrin). Many of the approaches which havebeen adopted to acquire fibrin antibodies have concentrated onimmunizing animals with soluble fibrin fragments and synthetic peptideswhich mimic exposed neoantigenic sites on fibrin. See Hui, K. Y., etal., Science 22, 1129-32 (1983), Scheefers-Borchel, V., et al., Proc.Natl. Acad. Sci USA, 82,7091-95(1985), Elms, M. J., et al., Thromb.Haemostas, 50, 591-94 (1983, and Kurdryk, B., et al., Mol. Immul., 21,89-94 (1984). However, it is believed that the binding site of suchantibodies is conserved during the fibrin degradation process and,therefore, such antibodies also can bind to fibrin degradation products.

The subject invention permits one to take a completely differentapproach and utilizes the intact fibrin antigen in conjunction with theenhanced immunological sensitivity of the antigen free (AF) animal toproduce a fibrin-specific monoclonal antibody. For the purpose of thesubject invention, a fibrin-specific monoclonal antibody binds to fibrinand not to fibrinogen, the fibrinogen degradation products or the fibrindegradation products.

5.2. MATERIALS AND METHODS 5.2.1. ANIMALS

Germfree BALB/cAnN mice were obtained from the germfree (GF) colonymaintained at the University of Wisconsin. The animals were transportedto our facility under GF conditions. The Antigen-Free (AF) colony wasinitiated by moving pregnant GF mice fed a natural ingredient diet L-485(See Pleasants, J. P., et al., J.Nutr. 116, 1949-1964 (1986)) to an AFisolator where the mice were immediately transferred to the chemicallydefined (CD) AF diet. Their offspring, which had never directlycontacted a Natural Ingredient (NI) diet, were weaned from maternal milkto CD diet and were designated the first AF generation. The AF mice weremated in pairs until the female was noticeably pregnant; then the malewas removed to ensure that the female would thereafter receive her fulldaily lipid supplement. Young were weaned at 24 days of age.

5.2.2. HOUSING

AF breeders were housed in pairs in one half of a standard polycarbonatemouse cage 28×17.8×12.7 cm. The bottom had been cut out and replacedwith a false bottom of mesh stainless steel. A longitudinal divider ofsheet stainless steel was bolted to the ends of the plastic cage,projecting enough above and beyond the cage to hold in place a lid ofstainless wire. This recessed lid, which normally fits inside a cage,was inverted to provide more adequate head room above the false bottom.Stainless steel collars of appropriate size were welded to the top ofthe lid to hold 60 mL diet bottles. Four stainless steel cups werewelded to the sides of the longitudinal divider at the ends, halfwaybetween the false bottom and the top. (A picture of the cages appears inPleasants, J. R., The germfree system for aging and immunity In: CRCHandbook of Immunology in Aging, (Kay, M. M. B. S. Makinodan, T., eds.),pp. 257-297, CRC Press, Boca Raton, Fla. (1981). The diet bottles wereof brown glass. Both diet and water bottles had plastic lids with holesdrilled in their centers. The bottles were filled and inverted in theircollars. The lipid supplement was measured daily into the stainlesssteel cups. A plastic pan was placed under each cage to receive wastes.

The filter paper which served both as bedding and as ingestible fiberwas Whatman ashless filter paper No. 41, purchased as clippings (SargentWelch). For bedding the paper was cut into strips. Uncut squares of thepaper were used for cleanup inside the isolator. The paper wasautoclaved for 25 min at 121° C., or was irradiated (4.5 Mrad) inplastic bags. All mice received enough paper to cover one end of thecage. It was replaced when it became wet, yellow or dirty.

The cages were maintained inside a 1.37×0.6×0.6M flexible isolator ofthe Trexler type (Trexler, P. C., Lab. Anim. Care, 13, 572-581 (1963)),using standard gnotobiotic technology (see Wostmann, B. S., Ed.,Gnotobiotes Standards and Guide Lines for the Breeding, Care andManagement of Laboratory Animals, National Research Council, NationalAcademy of Sciences, Washington, D.C.) The isolators were maintained ina room at 21° C. on a 12 h light dark schedule.

A 2.5 cm diameter Tygon tube 7.55 cm long was sealed to the top of theisolator and closed with vinyl stoppers at both top and bottom. Thisprovided an entry for sterile filtration of diet, water and oil.

5.2.3. DIET

Table 1 indicates both diet composition and the sequence for dissolvingthe ingredients in ultrafiltered Milli-Q water (Millipore, Mass.). Theamino acids and dextrose were Sigma tissue culture grade. Vitamins werealso from Sigma except for pure retinyl palmitate, kindly supplied byHoffman-La Roche, Inc. (Nutley, N.J.). The other reagents were Fishercertified or equivalent. The complete water soluble diet was filteredcold through an Amicon Diaflo TC3 ultrafilter using three Pm10 membranes150 mm in diameter (Amicon).

The ultrafilter membranes had a molecular weight cut off of 10,000daltons. The assembled ultrafilter apparatus was sterilized before useby passing a 0.15% sodium hypochlorite solution through it, followed bythorough washing. Ultrafiltered diet was stored at 4° C. in sterilereservoirs until needed. The diet was introduced into the AF isolatorusing a 0.2 um Nylon (MSI) filter in an autoclaved pressure filterholder with its delivery tube inserted into a No 6 Neoprene stopper. Forthis purpose, the upper vinyl stopper was removed from the Tygon tubesealed to the top of the isolator, and the interior of the tube wassprayed with a sterilizing solution of 2% peracetic acid containing 0.1%alkyl-aryl sulphonate. The filter holder stopper was inserted in placeof the upper stopper. After 20 minutes the lower stopper was removed(inside the isolator) and diet or water was filtered into the isolatorunder 20 psi of nitrogen.

The composition of the lipid supplement is given in Table 2. The soytriglycerides were a preparation made from those methyl esters whichvacuum distilled over a temperature range yielding the esters frompalmitate to linolenate. These esters were then transesterified withglycerol to form the mixed triglycerides. (Nu-Chek Prep, Elysian,Minn.). The fat-soluble vitamins were added to the triglyceride mixturebefore its filtration into the isolator at which time it was warmed to50° C. and filtered into the isolator by the same procedure used for thewater-soluble portion of the diet.

The lipid intake was a measured 0.375 ml/day. Increasing the lipidsupplement has greatly decreased the mortality rate of newborn mice. Theaverage litter size has also increased to that of conventionally rearedanimals. Lactating females received twice the normal amount of lipidsupplement.

                  TABLE 1    ______________________________________    Composition and preparation of    chemically defined diet L489E1Se                             Amount                             (grams/100 grams    Ingredient               of Ingredient)    ______________________________________    To 192 ml Milli-Q water 70° C. the following were added:    Leucine                  1.9    Phenylalanine            0.74    Isoleucine               1.08    Methionine               1.06    Tryptophan               0.37    Valine                   1.23    Asparagine               0.91    Arginine HCl             0.81    Threonine                0.74    Lysine HCl               1.77    Histidine HCl            0.74    The solution was cooled to 45° C. and the following    added:    Glycine                  0.59    Proline                  1.48    Serine                   1.33    Alanine                  0.59    Sodium glutamate         3.40    L-tyrosine ethyl ester HCI                             0.62    Ferrous gluconate        0.05    Salts 35D.sup.1          0.105    Sodiuin Selenite.sup.2   0.074    Solution cooled to 5° C. and the following added:    Calcium glycerophosphate 5.22    Magnesium glycerophosphate                             1.43    Calcium chloride 2H.sub.2 O                             0.185    Sodium chloride-Potassium Iodide                             0.086    (KI) mix (containing    680 mg KI)    Vitamin B mix 111E5.sup.3                             0.09    Vitamin B12.sup.4        1.20 mg    Choline chloride         0.31    Potassium acetate        1.85    To 108 ml Milli-Q water at 70° C.    D-Dextrose, anhydrous was added    Solutions cooled to 5° C. combined both for    ultrafiltration.    ______________________________________     .sup.1 Composition of salts 35D mixture given in Table 3     .sup.2 Added in addition to sodium selenite in Salts 35D     .sup.3 Composition of vitamin B mix 111E5 (see Table 4)     .sup.4 Added in addition to vitamin B12 in vitamin B mix 111E5

                  TABLE 2    ______________________________________    Composition of lipid supplement LADEK 69E6    Ingredient       Amount per daily dose* (0.375 ml)    ______________________________________    Purified soy triglycerides.sup.1                     0.33 g    Retinyl palmitate                     6.45 mg (11.7 I.U.)    Cholecalciferol  .0288 mg (1.15 I.U.)    2 ambo-alpha-tocopherol                     3.3 mg    2 ambo-alpha-tocopherol acetate                     6.6 mg    Phylloquinone    72.0 mg    ______________________________________     *Lactating mice received twice the normal adult dose.     .sup.1 Consisting of:     12% palmitate     1.5% stearate     24% oleate     55% linoleate     8% linolenate

                  TABLE 3    ______________________________________    Composition of the 35D Salts Mixture    Salt          Amount (per 300 ml of diet) (mg)    ______________________________________    Mn(acetate).sub.2 4H.sub.2 O                  55.4    ZuSO.sub.4 H.sub.2 O                  40.6    Cu(acetate).sub.2 H.sub.2 O                  3.7    Cr(acetate).sub.3 H.sub.2 O                  2.5    NaF           2.1    SnSO.sub.4 2H.sub.2 O                  0.37    (NH.sub.4).sub.6 Mo.sub.7 O.sub.24 4H.sub.2 O                  0.37    NiCl.sub.2 3H.sub.2 O                  0.37    Co(acetate).sub.2 4H.sub.2 O                  0.11    Na.sub.3 VO.sub.4                  0.22    Na.sub.2 SeO.sub.3                  0.096    ______________________________________

                  TABLE 4    ______________________________________    Composition of the Vitamin B Mixture 111E5    Ingredient     Amount (mg)/300 ml Diet)    ______________________________________    Thiamine HCl   1.23    Pyridoxine HCl 1.54    Biotin         0.25    Folic Acid     0.37    Vitamin B12    0.37    Riboflavin     1.85    Niacinamide    9.2    i-inositol     61.9    Calcium pantathenate                   12.3    ______________________________________

Water was Milli-Q ultrafiltered grade and was filtered into the isolatorin the same manner as the diet.

5.2.4. MICROBIOLOGICAL MONITORING

The antigen-free system was tested for microbial contamination accordingto guidelines set out in Wostmann, B. S. ed. (1970) GnotobioticsStandards and guidelines for the breeding care and management oflaboratory animals, National Research Council, National Academy ofSciences, Washington, D.C. Briefly, swabs wetted with diet and waterfrom inside the isolator were used to obtain fecal smears obtained freshfrom the mouse and from the accumulated waste under each cage. Smearswere also taken from the walls of the isolator particularly around theentry ports. Duplicate smears were always taken. One set was tested bydirect microscope examination for bacteria and fungi, using a gramstain. The second set of swabs was used for detection of microorganisms.Three weeks were allowed to elapse before a culture was considered to benegative.

Microbiological testing was performed approximately every two weeks or afew days after a new entry to the isolator had been made.

5.3. THE PRODUCTION OF MONOCLONAL ANTIBODIES USING ANTIGEN-FREE MICE

The antigen-free mice described hereinabove were used as the lymphocytedonor in the production of monoclonal antibodies. Solutions of allantigens were prepared under sterile conditions in a laminar flow hood.

The following protocol was adopted for the immunization of the AF mice.The antigen (25-50 micro g) was dissolved in sterile saline (100micro 1) and emulsified with an equal volume of Freund's CompleteAdjuvant (FCA). Interferon (1000 units) was added to the solution ofantigen prior to the preparation of the emulsion. Sterile syringes andneedles were used for all immunizations. The syringes were transferredto the AF isolator via the entry port where they were sterilized byspraying with a solution of peracetic acid (2%). Booster injections weregiven using the same amount of antigen and the replacement of FCA withFreund's Incomplete Adjuvant. A total of three booster immunizationswere given each at intervals of three weeks. The final boost (withoutadjuvant) was given 4-7 days prior to fusion. All immunizations weregiven intraperitoneally. The mice were removed from the isolator on theday of the fusion and were immediately sacrificed by CO₂ asphyxiationThe spleens were removed and the splenocytes fused with mouse myelomacells (NS1) using standard hybridoma technology.

5.4. USE OF THE ANTIGEN-FREE ANIMAL SYSTEM FOR THE PRODUCTION OF AFIBRIN-SPECIFIC MONOCLONAL ANTIBODY

The antigen-free system was used to generate a fibrin-specificmonoclonal antibody. The antibody is highly specific and does notrecognize fibrinogen, fibrin degradation products or fibrinogendegradation products. The hybridoma cell line was produced by fusion ofsplenocytes from antigen free BALB/c mice, immunized with human fibrin,and NS1 myeloma cells.

5.4.1. IMMUNIZATION SCHEDULE

Three eight week old female antigen free mice were immunized with 33micrograms of a human fibrin preparation. The preparation was a freezefracture sample of fibrin which was prepared as follows:

Human fibrinogen was converted to fibrin by thrombin and Factor XIIIa.The fibrin clot was then frozen in liquid nitrogen and reduced to anextremely fine powder by mechanical disruption. A dispersion of thefreeze fractured fibrin was made in saline to give a clear solution ofcrosslinked fibrin XL-Fn with a final concentration of 1 mg/mL. 33microL of this fibrin antigen was used to immunize the animals. Thevolume of the antigen solution was adjusted to 100 microL with sterilesaline and was then emulsified with FCA as described in the lastsection. Two booster immunizations were administered, at intervals ofthree weeks, using the same level of antigen in Freund's IncompleteAdjuvant. The final booster was given 4 days prior to the fusion. Thesame level of antigen was used and adjuvant was replaced with saline.

5.4.2. DETECTION AND DETERMINATION OF ANTIBODY

Qualitative and quantitative determinations of monoclonal antibody wereperformed using an enzyme linked immunosorbent assay (ELISA). The ELISAswere performed using human fibrin immobilized onto a 96 well PVC plate(Costar). The fibrin coated assay plates were prepared by incubating 100microL of a fibrinogen solution (Kabi, grade L) (50 micrograms/mLborate/saline buffer) overnight at 4° C. Unbound fibrinogen was removedby washing with PBS containing 0.05% Tween 80 (PBS-Tween). Thefibrinogen coated onto each plastic well was converted to fibrin byincubation with 100 microliters of a thrombin solution (10 NIH units/ml)containing 2mMCaCl₂ for 1 hour at 37° C. Standard calibration curves forthe antibody were constructed using a preparation of antibody which washomogeneous by SDS-PAGE.

To prevent non-specific binding, the fibrin coated plates were incubatedwith a 1% solution of BSA in PBS pH7.4. Antibody containing solutions(100 microL) were then added and incubated at 37° C. for 90 minutes.After each step in the procedure the wells were extensively washed withPBS-Tween. Bound antibody was detected by the addition of a 1000 folddilution of rabbit anti-mouse antibody conjugated to alkalinephosphatase (Sigma) diluted in PBS, 1% BSA pH8.0.

5.4.3. PRODUCTION OF HYBRIDOMAS-FUSION

The mice were sacrificed by CO₂ asphyxiation and a splenectomy performedimmediately. The spleen cells from the immunized mice were fused withthe fusagent polyethylene glycol 4000 (3000-3700). The cells wereincubated in HAT selection media in T flasks for 1 week. After this timethe cells were plated out into 5×96 well plates from which 93 wellsshowed growth. Of these, 19 wells were positive for the fibrin antigen.One of these clones, F492D8 (later renamed MH1), produced antibody whichrecognized the fibrin antigen but did not crossreact with fibrinogen.This particular clone, MH1, was recloned three times by limitingdilution with the tertiary cloning phase performed at 1 cell per well.Once the cell line was stabilized it was weaned onto a serum-free mediumthe cell line produces antibody at the level of approximately 7.5mg/liter.

5.4.4. PURIFICATION OF MONOCLONAL ANTIBODY

Before purification (4 Liter batches of) tissues culture supernatantswere centrifuged to remove cellular debris and filtered through a0.8microM nylon membrane to remove any residual particulate material.The hybridoma supernatant was concentrated at 4° C. to a volume of 500mL using a spiral wound ultrafiltration system employing a YM typemembrane (Amicon) with molecular weight cut of 30,000. Buffer exchangeto 20 mM 2(N-morpholine) ethane sulphonic acid (MES), pH6 (Buffer A) wasaccomplished by diafiltration according to the manufacturersinstructions. After further concentration to a final volume of 100 ml,the antibody solution was filtered through a 0.451 micron nylon membranebefore further purification. The concentrated antibody solution waspurified by liquid chromatography on a Waters HPLC chromatograph using a7.75 mm×10 cm ABx column (J. T. Baker, Phillipsburg, N.J.). The columnwas equilibrated with buffer A and the sample (100 ml) was applied at aflow rate of 1.0 ml/min. After extensive washing with buffer A theantibody was eluted from the column with a gradient from buffer A to100% buffer B (IM sodium acetate pH7) at 1 ml/min. Fractions (2ml) werecollected and those containing MAb (as determined by ELISA) were pooledand dialysed against phosphate buffer saline (PBS) (20 mM sodiumphosphate, 150 mM sodium chloride, pH7.4) and stored at -20° C. atconcentrations>1 mg/ml. The ABx column was regenerated by washing for 5minutes with 100% buffer B, followed by re-equilibration with 15 columnvolumes of buffer A.

5.5. DETERMINATION OF FIBRIN SPECIFICITY

Initial determination of fibrin specificity was achieved by screeninghybridoma supernatants separately on fibrin and fibrinogen coatedmicrotiter plates. Only those cell lines producing antibody that did notcrossreact with fibrinogen were accepted.

Further confirmation of fibrin specificity was determined utilizing acompetition assay with fibrinogen in solution, thereby confirming thatthe antibody does not recognize fibrinogen in solution.

The competition assay employed to ascertain the fibrin specificity ofthe antibody was performed as described for the ELISA assay hereinabovewith preincubation of the antibody with fibrinogen in solution. Briefly,hybridoma supernatant was incubated at 37° C. for 30 minutes withsolutions of fibrinogen at physiological concentrations (4 mg/ml)containing BSA (10 mg/ml) to prevent non specific binding of antibody tofibrinogen. The fibrinogen/antibody solution was then transferred towells of a microtiter plate which had been coated with fibrin.GlyProArgPro (GPRP) was added to the fibrinogen inhibitor to preventpossible polymerization of fibrinogen by residual thrombin in the fibrinwells. The assay is then performed as a conventional ELISA assay forantibody bound to an immobilized antigen. In all experiments to test thefibrin specificity of the MH1 antibody a second antibody 45J was used asa control. 45J crossreacts with fibrin and fibrinogen.

5.5.1. DETERMINATION OF CROSSREACTIVITY WITH FIBRINOGEN 5.5.1.1.IMMOBILIZED FIBRIN AND FIBRINOGEN

The cell line MH1 produces a murine monoclonal antibody whichcrossreacts with fibrin when it is immobilized on the surface of a PVCmicrotiter assay plate (table 5). In the same assay the antibody doesnot recognize fibrinogen immobilized on the plate. As the data on table5 indicate, there is a dramatic increase in immunoreactivity oncefibrinogen is converted to fibrin by thrombin, indicating clearly theexposure or formation of a neoepitope on the fibrin molecule. Thecontrol antibody, 45J, however clearly recognizes an epitope which isconserved when fibrinogen is converted to fibrin.

5.5.1.2. COMPETITION ASSAY WITH FIBRIN AND FIBRINOGEN

The fibrin specificity of the antibody, MH1 antibody, was furtherdemonstrated in a competition assay in which hybridoma supernatant waspreincubated with a fibrinogen solution (final concentration of 4 mg/ml)prior to an ELISA on fibrin coated wells. Since such a high level offibrinogen was used in this competition assay (×500 that of the antibodyconcentration) BSA (10 mg/ml final concentration) was added to themixture. The peptide GPRP was added to prevent fibrin polymerization byresidual thrombin on the fibrin coated wells. The results of this assayindicate that the MH1 antibody does not recognize fibrinogen in solution(Table 6).

Assuming 400 ng. of fibrin binds to each well of the microtiter assayplate, then the fibrinogen level used in this particular competitionassay represents a 1,000 fold excess over the bound fibrin antigen. Inaddition it represents a 400 fold excess of the antibody level in thetissue supernatant.

                  TABLE 5    ______________________________________    Crossreactivity of MH1 Antibody with    Fibrinogen and Fibrin Antigen                A.sub.405 nm /30 min.    MAb           Fibrin  Fibrinogen    ______________________________________    MH1           .90     .025    45J           1.65    1.50    ______________________________________

                  TABLE 6    ______________________________________    Crossreactivity of MH1 Antibody with Fibrin    in the Presence of Fibrinogen               A.sub.405nm /30 min.    MAb          +fibrinogen                           -fibrinogen    ______________________________________    MH1          1.24      1.27    45J          0.438     1.74    ______________________________________

5.5.2. DETERMINATION OF CROSSREACTIVITY WITH FIBRIN(OGEN) DEGRADATIONPRODUCTS

Fibrinogen degradation products (FDPs) were prepared by incubatingfibrinogen with plasmin at 37° C. for time periods ranging from 10minutes to 3 hours. At the desired time fibrinogenolysis was stopped bythe addition of Trasylol (100 Kallikrein inhibitor units/mL) and 20 mMepsilon amino caproic acid (EACA).

Crosslinked fibrin degradation products (XLFDPs) were prepared by theaddition of thrombin (4 NIH units/mL) to a fibrinogen solution (5 mg/mL)in Tris buffered saline (TBS, pH 7.4, 50 mM Tris HCl, 150 mM NaCl)containing 10 mM CaCl₂, plasminogen (0.25 mg/mL) and urokinase (50IU/mL). The mixture was incubated at 37° C. and the plasmin digestionterminated at different time intervals as described herein-above for theFDPs.

To determine crossreactivity of the antibody with fibrin degradationproducts and fibrinogen degradation products the appropriate degradationproducts were coated onto microtiter plates and ELISAs were performed byconventional methods. As the results in Table 7 indicate, the MH1antibody does not crossreact with any plasmin generated fibrinogendegradation products. As table 8 indicates, the antibody does not reactwith XL-fibrin degradation products. This observation also was made whenthe antibody was tested for crossreactivity by Western blottinganalysis.

The conclusion can be drawn that the antibody recognizes an epitope ofthe intact fibrin molecule which is not present or exposed on thesurface of the precursor molecule, fibrinogen. The epitope is apparentlydestroyed by plasmin digestion of crosslinked fibrin as the data intable 8 suggest.

Accordingly, the MH1 antibody is a fibrin-specific monoclonal antibodywhich can be defined as follows:

For the purpose of the subject invention a fibrin-specific monoclonalantibody is a monoclonal antibody that:

1. in a competition assay to measure crossreactivity with fibrin andfibrinogen, as described hereinabove, the monoclonal antibody has lessthan about 75%, and preferably less than about 10%, crossreactivity withfibrinogen when fibrinogen is utilized in a quantity of a 1,000 foldexcess as compared to fibrin,

2. in an assay to measure crossreactivity with crosslinked fibrin andfibrin degradation products, as described hereinabove, the reactivity ofthe monoclonal antibody with the fibrin that has been digested withplasmin for about three hours is less than about 50%, and preferablyless than about 40%, of the reactivity of the monoclonal antibody withfibrin at time zero, and

3. in an assay to measure crossreactivity with fibrinogen and fibrinogendegradation products, as described hereinabove, the reactivity of themonoclonal antibody with fibrinogen that has been digested with plasminfor about four hours is no greater than the reactivity of the monoclonalantibody with fibrinogen at time zero.

The MH1 antibody has been further characterized by determining itsaffinity for fibrin. The affinity was determined by Scatchard analysis(Frankel et al., Molecular Immunology, 16, 101-106(1979)) using125I-labelled MH1 antibody. The value obtained for the dissociationconstant K_(D) was 6.7×10⁻¹⁰ M. Such affinity is about 5,000 times thatof the affinity of t-PA for fibrin.

It has also been determined by Western Immunoblotting analysis that theMH1 antibody does not crossreact with the Aα, Bβ or gamma chains offibrinogen. Also, it has been determined by the same method of analysisthat the MH1 antibody does not crossreact with thrombin treated Aα or Bβchains of fibrinogen. (Thrombin treatment of fibrinogen results in therelease of fibrinopeptide A and fibrinopeptide B from the Aα chain andBβ chain, respectively, therefore forming the α chain and β chain offibrin.)

                  TABLE 7    ______________________________________    Crossreactivity of MH1 Antibody    with Fibrinogen Degradation Products    Plasmin Digestion Time                        A.sub.405 nm/30 min    (mins)              MH1    45J    ______________________________________    0                   0.15   1.037    10                  0.10   nm    20                  0.11   1.02    40                  0.11   0.410    60                  0.10   0.330    240                 0.09   0.300    ______________________________________

                  TABLE 8    ______________________________________    Crossreactivity of MH1 Antibody    with Fibrin Degradation Products    Plasmin Digestion Time                        A.sub.405 nm/30 min    (hours)             MH1    45J    ______________________________________    0                   0.890  1.40    3                   0.354  1.0    5                   0.310  0.77    ______________________________________

In addition, as the results in Table 7 indicate, the control antibody,45J antibody, binds to fibrinogen and does not crossreact withfibrinogen degradation products. Accordingly, such a monoclonal antibodyis a fibrinogen-specific monoclonal antibody and represents anotheraspect of the subject invention. The fibrinogen-specific monoclonalantibody can be utilized in any immunoassay that can be utilized todetermine plasma fibrinogen levels in vitro. The 45J antibody has beenfurther characterized in that it has been determined that the 45Jepitope is on the α chain of fibrinogen in the region from about aminoacid 206 to about 424 and most likely in the region from about aminoacid 207 to about 231. For the purpose of the subject invention, afibrinogen-specific monoclonal antibody is an antibody that in an assayto measure crossreactivity with fibrinogen and fibrinogen degradationproducts, as described hereinabove, the reactivity of the monoclonalantibody with fibrinogen that has been digested with plasmin for about40 minutes is less than about 50% of the reactivity of the monoclonalantibody with fibrinogen at time zero.

The 45J hybridoma was made by conventional techniques utilizing aconventional Balb/c mouse wherein the mouse was immunized with fibrin.However, fibrinogen also can be utilized as the antigen.

5.5.3. DETERMINATION OF CROSS REACTIVITY WITH NONCROSSLINKED FIBRIN ANDNONCROSSLINKED FIBRIN CLOTS

It has been demonstrated by ELISA that the antibody MH1 crossreacts withnot only crosslinked fibrin (XLFn) but also noncrosslinked fibrin(NONXLFn). The ELISA was performed as follows:

1. 96 well microtiter assay plates were coated with 100 ul of afibrinogen solution (50 ug/mL in borate (pH8.5) saline buffer) at 4° C.overnight.

2. Crosslinked fibrin was formed in the fibrinogen coated wells byincubation for 1 hour at 37° C. with a thrombin solution (10 NIHunits/ml) in Tris buffered saline (TBS, pH 7.4, 50 mM Tris HCl, 150 mMNaCl), containing 2 mM CaCl₂ and 10 mM cysteine.

3. Noncrosslinked fibrin was formed in the fibrinogen coated wells byincubation for 1 hour at 37° C. with a thrombin solution (10 NIHunits/ml) in phosphate buffer (pH 6.1) containing EDTA to a finalconcentration of 0.0125M.

4. The bound antibody was determined by incubation with an antimousealkaline phosphatase conjugate. Bound conjugate was determined by theaddition of an alkaline phosphatase substrate and the resultantcolorimetric reaction monitored at 405 nM in an automatic plate reader.

The results of the assay are shown in Table 9 and it can be concludedthat the antibody crossreactivity with crosslinked fibrin is greaterthan it is with noncrosslinked fibrin. The simplest explanation beingthat the covalent crosslinking present in the crosslinked polymericstructure serves to lock or freeze the conformation which the antibodyrecognizes. In the noncrosslinked species the conformation, although itis formed, it is not stabilized by covalent bonding of the polymer.

It has also been demonstrated that the antibody crossreacts with bothcrosslinked and noncrosslinked clots, formed in vitro. Crosslinkedfibrin was prepared by incubating a fibrinogen solution (In Tris (50 mM,pH7.4) saline, containing CaCl₂ (2 mM) and cysteine (10 mM)) withthrombin (10 NIH units/ml) for 3 hours at 37° C. The non-crosslinedfibrin was formed by incubating a fibrinogen solution (3mg/ml) inphosphate buffer (pH6.1) containing EDTA to a final concentration of0.0125M with a thrombin solution (10 NIH units/ml) for 3 hours at 37° C.After formation of the clots, they were washed and incubated at 37° C.with 400 ul of a 1% BSA solution containing ¹²⁵ Iodine-labelled MH1antibody. Small aliquots of solution were removed at different timepoints and the amount of antibody uptake was determined by counting theplasma in a gamma counter.

Table 10 shows the uptake of antibody by both the noncrosslinked andcrosslinked clots.

                  TABLE 9    ______________________________________    Crossreactivity of MH1 Antibody with Crosslinked and    Noncrosslinked Fibrin    Antibody      Crosslinked                            Noncrosslinked    Concentration Fibrin    Fibrin    (ng./ml.)     (Absorbance at 405 nanometers)    ______________________________________    100.000       0.764     0.409    50.000        0.460     0.319    25.000        0.305     0.154    12.500        0.174     0.074    6.250         0.069     0.000    ______________________________________

                  TABLE 10    ______________________________________    Crossreactivity of MH1 Antibody with Crosslinked    and Noncrosslinked Fibrin Clots    Fibrin Clot               % Uptake of Labelled Antibody After 8 Hours    ______________________________________    Crosslinked               79%    Noncrosslinked               76%    ______________________________________

5.6. DEPOSIT OF HYBRIDOMA

MH1 and 45J were deposited in the American Type Culture Collection(ATCC) on Jun. 9, 1988 and given accession number HB 9739 and HB 9740,respectively. The ATCC is located at 12301 Parklawn Drive, Rockville,Md. 20852. MH1 antibody is an IgG₁ antibody with a kappa light chain andit has been observed that the MH1 antibody crossreacts with not onlyhuman fibrin but also rabbit fibrin.

The subject invention is not intended to be limited in scope to thehybridomas deposited but they are intended as a single illustration ofhybridomas that produced a fibrin-specific monoclonal antibody and afibrinogen-specific monoclonal antibody, as defined herein. Any cellline that is functionally equivalent is within the scope of the subjectinvention. By the term "functionally equivalent" it is meant that anantibody is capable of competing with the MH1 antibody or 45J antibodyin binding to the epitope of fibrin to which the MH1 antibody binds orto the epitope of fibrinogen to which the 45J antibody binds,respectively. In addition, such term includes fibrin-specific monoclonalantibodies and fibrinogen-specific monoclonal antibodies, as definedherein, that bind to an epitope different from that which the MH1antibody binds and the 45J antibody binds, respectively.

5.7. IN VIVO DIAGNOSTIC AND THERAPEUTIC USES FOR FIBRIN-SPECIFICMONOCLONAL ANTIBODIES 5.7.1. CLOT/VASCULAR DISEASE LOCALIZATION

The fibrin-specific monoclonal antibodies of this invention are capableof targeting fibrin clots or aggregation of fibrin in vivo. They can,therefore, be used in humans for localization of possible tissue orvascular damage and for monitoring of vascular diseases. Afibrin-specific monoclonal antibody is particularly preferred for thisuse because such monoclonal antibody will not bind to fibrinogen, fibrindegradation products and fibrinogen degradation products, therebyreducing background, which permits one to more precisely localize thefibrin clot or aggregation of fibrin.

For this application, it is preferable to use purified monoclonalantibodies. Preferably, purification may be accomplished by HPLCmethods. Purification of monoclonal antibodies for human administrationmay also be accomplished by ammonium sulfate or sodium sulfateprecipitation followed by dialysis against saline and filtrationsterilization. Alternatively, immunoaffinity chromatography techniquesmay be used to purify the monoclonal antibodies.

The purified monoclonal antibodies can be labelled with radioactivecompounds, for example ¹²³ I, ¹²⁵ I, ¹³¹ I, ⁹⁹ mTc, ¹¹¹ In, andadministered to a patient intravenously. The antibody also can belabelled with a magnetic probe. NMR can then be utilized to pinpoint theclot. After localization of the antibodies at the clot or fibrinaggregation they can be detected by emission tomographical andradionuclear scanning techniques thereby pinpointing the location of,for example, the thrombus or fibrin encapsulated tumor.

By way of illustration, the purified monoclonal antibody is suspended inan appropriate carrier, e.g., saline, with or without human albumin, atan appropriate dosage and is administered to a patient. The monclonalantibodies are preferably administered intravenously, e.g., bycontinuous intravenous infusion over several hours.

5.7.2. TREATMENT OF VASCULAR DISEASES WITH MONOCLONAL ANTIBODYCONJUGATES

The monoclonal antibodies of this invention can be used in conjunctionwith a broad range of pharmaceutical agents such as cytotoxic reagentsand thrombolytic reagents, e.g. t-PA, urokinase streptokinase, and otherproteases that are capable of lysing fibrin. Such use is particularlypreferred because the fibrin-specific monoclonal antibodies of thesubject invention permit a very efficient use of such reagents becausenone of the reagent will be lost by binding to fibrinogen, fibrindegradation products or fibrinogen degradation products. For variousreviews on the subject, see Bale et al., 1980, Cancer Research,40:2965-297; Ghose and Blair, 1978, J. Natl. Cancer Inst.,61(3):657-676; Gregoriadis, 1977, Nature, 265:407-411; Gregoriadis, 1980Pharmac. Ther., 10:103-108; and Trouet et al., 1980, Recent ResultsCancer Res., 75:229-235.

The methods used for binding these agents to the monoclonal antibodymolecule can involve either non-covalent or covalent linkages. Sincenon-covalent bonds are more likely to be broken before the antibodycomplex reaches the target site, covalent linkages are preferred. Forinstance, a carbodiimide bond can be formed between the carboxy groupsof the pharmaceutical agent and the amino groups of the antibodymolecule. Bifunctional agents such as dialdehydes or imidoesters can beused to link the amino group of a drug to amino groups of the antibodymolecule. The Schiff base reaction can be used to link drugs to antibodymolecules. This method involves the periodate oxidation of a drug orcytotoxic agent that contains a glycol or hydroxy group, thus forming analdehyde which is then reacted with the antibody molecule. Attachmentoccurs via formation of a Schiff base with amino groups of the antibodymolecule. Additionally, drugs with reactive sulfhydryl groups have beencoupled to antibody molecules.

6. IN VITRO DETECTION AND MEASUREMENT OF SOLUBLE FIBRIN 6.1 BACKGROUND

As described above, see Section 5.1, the hemostatic mechanism involves acomplex sequence of reactions, by which fibrinogen is ultimatelyconverted by thrombin to fibrin. The end result of these reactions isthe formation of a thrombus (blood clot). The sequence of reactions maybe simply represented by a three step process as follows:

    ______________________________________    Thrombin    ______________________________________    Step 1 - Proteolysis: Fibrinogen →    Fibrin Monomers + Fibrinopeptides A and B    Step 2 - Polymerization: Fibrin monomers ⃡    Soluble Fibrin Polymers    Step 3 - Clotting: Soluble Fibrin Polymers →    Fibrin Clot    ______________________________________

Fibrinogen is composed of three pairs of non-identical polypeptidechains: Aα, Bβ and γ. See L. Stryer, Biochemistry, Third Edition p. 249,W. H. Freeman and Company New York (1988). In the initial step, wherebyfibrinogen is converted to fibrin, shown above as step 1, fibrinogen iscleaved by thrombin to release fibrinopeptide A from the amino-terminalends of the two fibrinogen Aα-chains. The resultant monomer is the DesAAfibrin monomer. As also shown above in step 1, simultaneously, but moreslowly, thrombin also cleaves fibrinopeptide B from the amino-terminalends of the two fibrinogen Bβ-chains. As a result of the fibrinopeptiderelease, new amino-terminals are exposed on the fibrin a and β chains.As depicted above, the molecules formed in step 1 are fibrinopeptide A,fibrinopeptide B, and the fibrin monomers DesAA fibrin monomer, andDesAABB fibrin monomer, shown above as "fibrin monomers". See W.Nieuwenhuizen, Blood Coagulation and Fibrinolysis, 4:93-96 (1993). Asshown above in step 2, the fibrin monomers then form both non-covalent(non-crosslinked) and covalent (crosslinked) polymers to form solublefibrin polymers. As shown above in step 3, the soluble fibrin polymersthen form the fibrin clot.

Soluble fibrin is defined as any molecular species originating fromfibrinogen or fibrin that can lead to fibrin polymer formation or anyfibrin(ogen) derived molecular species which has a molecular weightgreater than the molecular weight of native fibrinogen, and ismaintained in solution in blood. Non-crosslinked and crosslinked DesAABBfibrin polymers, formed in step 2 above, are two of the several speciesof soluble fibrin and are also two species of soluble fibrin polymer.Additionally, the term soluble fibrin includes various other species;for example, DesAA fibrin polymers, complexes formed by interactionsbetween fibrin monomers (either DesAA or DesAABB fibrin monomers) andthe fibrinogen degradation products X, Y, D and E (see Section 5.1 abovefor a description of these degradation products) and also, for example,DesAA and DesAABB monomers in complex with fibrinogen, seeNieuwenhuizen, pp. 93-94.

Soluble fibrin polymers are the immediate precursors of the insolublefibrin, i.e., the clot, and consequently the plasma levels of thesoluble fibrin polymers are believed to be elevated in individuals withimpending or existing thrombosis (intravascular blood clot formation).The detection and measurement of the amount of these polymers in blood,in particular the DesAABB soluble fibrin polymers would therefore, beuseful as an indication of incipient blood clot formation. See Bang andChang, pp. 119-121, and Nieuwenhuizen, p. 94, Marder et al. U.S. Pat.No. 5,206,140.

Certain species of soluble fibrin have previously been detected ormeasured and detected using a variety of methods including, for example,measurement of fibrinopeptide A, measurement by using antibodies to theAα and γ epitopes exposed upon conversion of fibrinogen to fibrin,Nieuwenhuizen, 94-96, and measurement of D-dimers, Marder et al. U.S.Pat. No. 5,206,140. Other methods used to detect or measure and detectsoluble fibrin include, measurement of agglutination of erythrocytescoated with fibrin in the presence of soluble fibrin, gel exclusionchromatography, rate enhancement of plasminogen activation by theplasminogen activator t-PA, see Nieuwenhuizen, at p. 94, ethanol orprotamine sulfate gelation, N-terminal analysis of fibrinogen fractionspurified from plasma, incorporation of ¹⁴ C-labeled glycine-ethyl esterand agarose gel chromatography, see Bang and Chang at pp. 111-118. Noneof these tests detects and measures specifically both solublecrosslinked and soluble non-crosslinked fibrin polymers.

6.2 IN VITRO ASSAY FOR SOLUBLE CROSSLINKED FIBRIN POLYMERS AND SOLUBLENON-CROSSLINKED FIBRIN POLYMERS

The present invention is based upon the discovery that it is possible toprovide an in vitro assay to detect and measure the amount of solublecrosslinked DesAABB fibrin polymers and soluble non-crosslinked DesAABBfibrin polymers, which polymers are composed of DesAABB fibrin monomers,in an assay system wherein there is no detection of (a) fibrinogen, (b)fibrinogen degradation products, (c) DesAA fibrin monomers, (d) DesAAfibrin polymers, (e) DesAABB fibrin monomers, (f) crosslinked fibrinogen(Factor Xllla treated fibrinogen), (g) DesAA fibrin monomer-fibrinogencomplexes and (h) fibrin degradation products ("species (a)-(h)"). Thefibrinogen degradation products and the fibrin degradation products arethose generated by plasmin digestion of fibrinogen or fibrin asdescribed in Section 5.5.2 above.

It is believed that the above described assay is particularly useful inthe clinical diagnosis of conditions characterized by thrombosis.

The assay may be carried out utilizing any suitable sample of body fluidbut is preferably done utilizing as a sample mammalian blood. Suitablemammals include for example rabbits, monkeys, and humans with humansbeing most preferred.

In such in vitro assays, any means, known or to be developed, ofdetecting soluble crosslinked DesAABB fibrin polymers and solublenon-crosslinked DesAABB fibrin polymers which means does not detectspecies (a)-(h) can be utilized. Once detected, the soluble crosslinkedDesAABB fibrin polymers and soluble non-crosslinked DesAABB fibrinpolymers in a sample can be measured either by comparison to a controlsample or by use of standards having known amounts of solublecrosslinked DesAABB fibrin polymers and soluble non-crosslinked DesAABBfibrin polymers which standards contain none of species (a)-(h).

It is to be noted that a means of detection is any means by which one isable to determine the presence of the material of interest in a sample.A preferred means of detection is an antibody to the soluble crosslinkedDesAABB fibrin polymers and the soluble non-crosslinked DesAABB fibrinpolymers, which antibody does not cross-react with (a) fibrinogen, (b)fibrinogen degradation products, (c) DesAA fibrin monomers, (d) DesAAfibrin polymers, (e) DesAABB monomers, (f) crosslinked fibrinogen, (g)DesAA fibrin monomer-fibrinogen complexes and (h) fibrin degradationproducts. Said antibody can be used to detect and measure the amount ofsoluble crosslinked DesAABB fibrin polymer and soluble non-crosslinkedDesAABB fibrin polymer in a sample. Such antibodies include polyclonalor monoclonal antibodies, preferably monoclonal. The antibody can bederived from any species. Preferably, however, the antibody is of humanor murine, or rabbit origin. In addition, such antibodies include, butare not limited to chimeric antibodies, single chain antibodies, and Fabfragments.

An example of such antibody is the monoclonal antibody MH1, described insections 5.5.1-5.5.3, and also section 5.6 above. It is believed thatMHI, in addition to having the above described binding characteristics,also does not bind to the fibrinogen-fibrin degradation product complex.This characteristic of MHI may enhance its efficacy as a means ofdetecting and measuring soluble crosslinked and soluble non-crosslinkedDesAABB fibrin polymers.

It is to be noted that two or more antibodies can be used as the meansof detection. Thus, antibodies with different specificities can be usedin combination to detect and measure soluble crosslinked DesAABB fibrinpolymers and soluble non-crosslinked DesAABB fibrin polymers in asample, wherein for example, neither antibody alone is able to form acomplex with both soluble crosslinked DesAABB fibrin polymers andsoluble non-crosslinked DesAABB fibrin polymers, whereas the two or moreantibodies can form such complexes. Of course, no one antibody cancrossreact with species (a)-(h).

It is also to be noted that when antibodies are used it may be necessaryto utilize a species specific antibody.

For the production of antibodies, various host animals can be immunizedby injection with soluble crosslinked DesAABB fibrin polymers andsoluble non-crosslinked DesAABB fibrin polymers as the antigen,including but not limited to rabbits, mice, rats, etc. Such polymers canbe selectively isolated using, for example, the MH1 antibody, coupled toa Sepharose MH1 antibody affinity column. Such polymers can be preparedas follows: Soluble fibrin can first be prepared in vitro by addition oflow levels of thrombin to citrated plasma. After quenching of thethrombin activity by addition of a potent thrombin inhibitor such ashirudin, the plasma samples may be applied to a Sepharose-MH1 column.Such MH1 column may be prepared by the coupling of MH1 antibody tocyanogen bromide activated Sepharose 4B (Pharmacia) as is known in theart. See Pharmacia product insert for methods for preparing anantibody-Sepharose 4B column using preactivated Sepharose 4B. Thesoluble fibrin is initially applied to the Sepharose-MH1 column in thepresence of phosphate buffered saline (PBS). After binding to theSepharose-MH1 column, in the presence of (PBS) the soluble fibrinbinding to the MH1-Sepharose may be eluted using a solution of sodiumthiocyanate. After dialysis against PBS, the soluble fibrin polymerwhich has been isolated may be stored at -70° C.

Various adjuvants can be used in conjunction with the isolated polymersto increase the immunological response, depending on the host species,including but not limited to Freund's (complete and incomplete), mineralgels such as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum.

Monoclonal antibodies specific for soluble crosslinked and solublenon-crosslinked DesAABB fibrin polymers which antibodies do not crossreact with species (a)-(g) can be prepared by using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include but are not limited to the hybridomatechnique originally described by Kohler and Milstein, (Nature, 1975,256:495-497), the more recent human B-cell hybridoma technique (Kosboret al., 1983, Immunology Today, 4:72) and the EBV-hybridoma technique(Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96). In an additional embodiment of the inventionmonoclonal antibodies can be produced in germ-free animals utilizingrecent technology, see sections 5.2-5.4. An example of the production ofsuch antibody is the production of MH1 described above in sections5.3-5.6.

According to the invention, human antibodies can be used and can beobtained by using human hybridomas (Cote at al., 1983, Proc. Natl. Acad.Sci., 80:2026-2030) or by transforming human B cells with EBV virus invitro (Cole et al., 1985, in, Monoclonal Antibodies and Cancer Therapy,Alan R. Liss, pp. 77-96). In fact, according to the invention,techniques developed for the production of "chimeric antibodies"(Morrison et al., 1984, Proc. Natl. Acad. Sci., 81:6851-6855; Neubergeret al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature,314:452-454) by splicing the genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used; suchantibodies are within the scope of this invention.

According to the invention, techniques described for the production ofsingle chain antibodies (U.S. Pat. No. 4,946,778) can be adapted toproduce specific single chain antibodies.

An additional embodiment of the invention utilizes the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,1989, Science, 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity to solublecrosslinked and soluble non-crosslinked DesAABB fibrin polymers.

Antibody fragments which contain specific binding sites for solublecrosslinked and soluble non-crosslinked DesAABB fibrin polymers may begenerated by known techniques. For example, such fragments include butare not limited to: the F(ab')₂ fragments which can be produced bypepsin digestion of the antibody molecule and the Fab fragments whichcan be generated by reducing the disulfide bridges of the F(ab')₂fragments.

The antibodies of the present invention may be used in any immunoassaysystem known in the art including, but not limited to:radioimmunoassays, enzyme-linked immunosorbent assays, "sandwich"assays, precipitin reactions, gel diffusion immunodiffusion assays,agglutination assays, fluorescent immunoassays, protein A immunoassaysand immunoelectrophoresis assays, to name but a few. U.S. Pat. No.4,629,783 and patents cited therein also describe suitable assays.

The antibodies may be used as the basic reagents in a number ofdifferent immunoassays to determine the presence of the solublecrosslinked and soluble non-crosslinked DesAABB fibrin polymers in asample of blood or other body fluid. Generally speaking, the antibodiescan be employed in any type of immunoassay, whether qualitative orquantitative. This includes both the two-site sandwich assay and thesingle site immunoassay of the non-competitive type, as well astraditional competitive binding assays.

Particularly preferred, for ease of detection, and its quantitativenature, is the sandwich or double antibody assay, of which a number ofvariations exist, all of which are intended to be encompassed by thepresent invention.

For example, in a typical forward sandwich assay, unlabeled antibody isimmobilized on a solid substrate, e.g., microtitre plate wells, and thesample to be tested is brought into contact with the bound molecule.After a suitable period of incubation, for a period of time sufficientto allow formation of an antibody-antigen binary complex, a secondantibody, labelled with a reporter molecule capable of inducing adetectable signal, is then added and incubation is continued allowingsufficient time for binding with the antigen at a different site and theformation of a ternary complex of antibody-antigen-labeled antibody. Anyunreacted material is washed away, and the presence of the antigen isdetermined by observation of a signal, which may be quantitated bycomparison with control samples (standards) containing known amounts ofantigen. Variations on the forward sandwich assay include thesimultaneous assay, in which both sample and antibody are addedsimultaneously to the bound antibody, or a reverse sandwich assay inwhich the labelled antibody and sample to be tested are first combined,incubated and added to the unlabelled surface bound antibody. Thesetechniques are well known to those skilled in the art, and thepossibility of minor variations will be readily apparent. As usedherein, "sandwich assay" is intended to encompass all variations on thebasic two-site technique.

As a more specific example, in a typical forward sandwich assay, aprimary antibody is either covalently or passively bound to a solidsupport. The solid surface is usually glass or a polymer, the mostcommonly used polymers being cellulose, polyacrylamide, nylon,polystyrene, polyvinylchloride or polypropylene. The solid supports maybe in the form of tubes, beads, discs or microplates, or any othersurfaces suitable for conducting an immunoassay. The binding processesare well known in the art. Following binding, the solid phase-antibodycomplex is washed in preparation for the test sample. An aliquot of thebody fluid to be tested is then added to the solid phase complex andincubated for a period of time sufficient to allow binding of anysoluble non-crosslinked DesAABB fibrin polymer and soluble crosslinkedDesAABB fibrin polymer present to the antibody specific for the aboveproteins. The second antibody is then added to the solid phase complexand incubated for an additional period of time sufficient to allow thesecond antibody to bind to the primary antibody-antigen solid phasecomplex. The second antibody is linked to a reporter molecule, thevisible signal of which is used to indicate the binding of the secondantibody to any antigen in the sample. By "reporter molecule", as usedin the present specification is meant a molecule which by its chemicalnature, provides an analytically detectable signal which allows thedetection of antigen-bound antibody. Detection must be at leastrelatively quantifiable, to allow determination of the amount of antigenin the sample, this may be calculated in absolute terms, or may be donein comparison with a standard (or series of standards) containing aknown normal level of antigen.

The most commonly used reporter molecules in this type of assay areeither enzymes or fluorophores. In the case of an enzyme immunoassay anenzyme is conjugated to the second antibody, often by means ofglutaraldehyde or periodate. As will be readily recognized, however, awide variety of different conjugation techniques exist, which are wellknown to the skilled artisan. Commonly used enzymes include horseradishperoxidase, glucose oxidase, β-galactosidase and alkaline phosphatase,among others. The substrates to be used with the specific enzymes aregenerally chosen for the production, upon hydrolysis by thecorresponding enzyme, of a detectable color change. For example,p-nitrophenyl phosphate is suitable for use with alkaline phosphataseconjugates; for peroxidase conjugates, 1,2-phenylenediamine or toluidineare commonly used. It is also possible to employ fluorogenic substrates,which yield a fluorescent product rather than the chromogenic substratesnoted above. In all cases, the enzyme-labelled antibody is added to thefirst antibody complex and allowed to bind to the complex, then theexcess reagent is washed away. A solution containing the appropriatesubstrate is then added to the tertiary complex ofantibody-antigen-labelled antibody. The substrate reacts with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anevaluation of the amount of antigen which is present in the serumsample.

Alternately, fluorescent compounds, such as fluorescein or rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labelled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.As in the enzyme immunoassay (EIA), the fluorescent-labelled antibody isallowed to bind to the first antigen-complex. After washing the unboundreagent, the remaining ternary complex is then exposed to light of theappropriate wavelength, and the fluorescence observed indicates thepresence of the antigen. Immunofluorescence and EIA techniques are bothvery well established in the art and are particularly preferred for thepresent method. However, other reporter molecules, such asradioisotopes, chemiluminescent or bioluminescent molecules may also beemployed. It will be readily apparent to the skilled artisan how to varythe procedure to suit the required use.

Alternatively, the sample to be tested, either mammalian blood or otherbody fluid containing the soluble non-crosslinked fibrin DesAABB polymerand the soluble crosslinked DesAABB fibrin polymer may be used in asingle site immunoassay wherein it is adhered to a solid substrateeither covalently or noncovalently. An unlabeled antibody is broughtinto contact with the sample bound on the solid substrate. After asuitable period of incubation, for a period of time sufficient to allowformation of an antibody-antigen binary complex a second antibody,labelled with a reporter molecule capable of inducing a detectablesignal, is then added and incubation is continued allowing sufficienttime for the formation of a ternary complex of antigen-antibody-labeledantibody. For the single site immunoassay, the second antibody may be ageneral antibody (i.e., zenogeneic antibody to immunoglobulin,particularly anti-(IgM and IgG) linked to a reporter molecule) that iscapable of binding an antibody that is specific for the solublecrosslinked fibrin polymer and the soluble non-crosslinked fibrinpolymer.

The detection and measurement of soluble non-crosslinked DesAABB fibrinpolymers and soluble crosslinked DesAABB fibrin polymers in vitro isparticularly useful when such detection and measurement is done, usingthe plasma of patients, to obtain an indication of an impending orexisting thrombotic event, said event being due to an impending orexisting thrombosis. See, e.g., W. Nieuwenhuizen, p. 94, Bang and Chang,pp. 109-122, and Marder et al. U.S. Pat. No. 5,206,140. Such eventsinclude, for example, deep vein thrombosis ("DVT"), a condition whicharises as a result of blood clot formation in the deep veins of the leg;pulmonary embolism (PE), which arises when a thrombus (blood clot)becomes dislodged from the deep veins and embolizes to the pulmonaryvasculature; disseminated intravascular coagulation, which arises as aresult of systemic activation of the blood clotting cascade (e.g., inbacterial infection); myocardial infarction (MI), which arises as aresult of a thrombus occluding the coronary arteries which supply bloodto the heart muscle; stroke; and intracardiac thrombi formed as a resultof atrial fibrillation. The type of thrombotic event can be diagnosed byuse of the assay for detection and measurement of solublenon-crosslinked DesAABB fibrin polymers and soluble crosslinked DesAABBfibrin polymers in combination with observation of other patientsymptoms. The symptoms utilized are those which would be commonlyutilized in clinical diagnosis of an impending thrombotic event. Forexample, said detection and measurement of soluble DesAABB fibrinpolymers is particularly useful as a means of differentially diagnosingpatients with chest pain due to impending MI from patients with chestpain due to other conditions.

6.3 EXAMPLES OF MATERIALS & METHODS 6.3.1. PROTEINS

Bovine Serum Albumin and Thrombin were both purchased from ICN (CostaMesa, Calif.). Hirudin, horseraddish peroxidase and o-phenylenediaminedihydrochloride substrate tablets were all purchased from Sigma ChemicalCo. (St. Louis, Mo.). Human fibrinogen (grade L) was purchased fromHelena Laboratories (Beaumont, Tex.). Fibrinogen was further purified byammonium sulphate precipitation as described by Holm et al., Thromb.Res, 37:165-176 (1985). The anti-fibrin monoclonal antibody, MH1, usedin the assay system was produced as described, section 5 above. Theantifibrinogen monoclonal antibody, 45J, was produced as described,section 5 above.

DesAABB fibrin monomer was prepared by dissolving non-crosslinked fibrinpolymer in a solution of 50 mM sodium acetate buffer (pH 5.3) containingsodium bromideim.

Crosslinked soluble DesAABB fibrin polymers were produced by incubationof fibrinogen or citrated plasma with a low level of thrombin (0.025units/ml) for 7-8 minutes. The reaction mixture also contained FactorXIIIa. The reaction was quenched by the addition of hirudin to thereaction mixture, final concentration 10 ATU/ml).

Non-crosslinked soluble DesAABB fibrin polymers were produced byincubating citrated plasma or fibrinogen with a solution of thrombincontaining EDTA (12 mM final concentration) for 7-8 minutes at 37° C.The reaction was quenched by the addition of excess hirudin to thereaction mixture, (final concentration 10 ATU/ml).

6.3.2. MH1 AND 45J HORSERADISH PEROXIDASE CONJUGATE (adapted from Wilsonand Nakane, Immunofluorescence and Related Staining Techniques, Knapp,et al. (eds), pp. 215-224, Elsevier/North Holland Biomedical Press,Amsterdam (1978))

Horseradish peroxidase (HRP) (RZ>3) (10 mg) was dissolved in 1 ml ofdistilled water. A freshly prepared solution of 0.1M sodium periodate(0.4 ml) was added and mixed gently for 20 minutes. The solution wasthen transferred to a sodium acetate buffer (1% mM, pH 4.4) at 4° C.using a Centricon 10 micro-concentrator.

The pH of the solution was raised to 9.5 by adding 40 μl of 0.2M sodiumcarbonate-bicarbonate buffer (pH 9.5). MH1 antibody (20 mg) in 2.0 ml of0.01M sodium carbonate-bicarbonate buffer (pH 9.5) was then addedwithout delay. This solution was mixed gently at room temperature for 2hours after which 0.2 ml of a freshly prepared sodium borohydridesolution (4 mg/ml) was added. The mixture was allowed to stand for 2hours at 4° C.

Finally the antibody HRP conjugate was dialyzed against 5×4 liters ofPBS at 2°-8° C.

The conjugate was stored in a brown container at 2°-8° C.

6.3.3. BLOOD SAMPLES

Blood samples were acquired with consent from both healthy volunteersand from emergency room patients who presented clinical symptoms of MI,e.g., severe chest pain. The blood samples were collected into sodiumcitrate using vacutainers (Becton Dickinson). Plasma was separated bycentrifugation at 2400 RPM for 15 minutes. The plasma was either usedimmediately or stored frozen at -70° C.

6.3.4. ELISA PROCEDURE FOR MEASURING SOLUBLE FIBRIN

Soluble fibrin was detected in fresh or frozen plasma samples using asandwich type enzyme-linked immunoassay system. The capture antibody inthe current invention was the antifibrin MH1 antibody. The detection (ortag antibody) was a HRP conjugate of the same antibody. Alternatively, aHRP conjugate of the antifibrinogen antibody 45J, could be employed asthe tagging antibody.

96 well polyvinyl chloride (PVC) microtitre plates (Costar Cambridge,Mass.) were coated with the monoclonal antibody MH1 by incubation of a100 μl solution of the Mab (at 50 μg/mL) in coating buffer (sodiumborate, ph 8.5) for 12-16 hours at 4° C. Unbound antibody was removedfrom the plates by washing the wells three times with a PBS-Tweensolution. The MH1-coated wells were postcoated with BSA by incubating a200 μl solution of BSA (1%) in PBS (PBS-BSA) for 1 hour at 37° C.Unbound BSA was removed by inverting the plates and tapping gently ontoa paper towel. Citrated samples (50 μl) containing soluble fibrin wereincubated on the MH1-BSA blocked wells for 30 minutes at 37° C. Unboundmaterial was removed by inversion and gentle tapping of the microtitreplates. The wells were then washed three times with PBS-Tween. Boundsoluble fibrin was detected by first incubating 100 μl of PBS-BSAsolution of the MH1-HRP conjugate (or a HRP conjugate of anantifibrin(ogen) antibody, 45J) in the wells for 30 minutes at 37° C.Unbound conjugate was removed by inverting the plate and washing thewells 3 times with PBS-Tween. The bound conjugate was detected byaddition of 100 μl of O-phenylenediamine dihydrochloride solution for 10minutes at room temperature. The substrate solution was prepared bydissolving a table of O-phenylenediamine dihydrochloride in a sodiumcitrate solution containing H₂ O₂.

The calorimetric reaction was quenched after 10 minutes by addition of a25 μL solution of H₂ SO₄ (1M). The absorbance of the solution in eachwell was determined at 490 nm in a Thermomax microtitre plate reader(Molecular Devices, Menlo Park, Calif.)

6.3.5 PREPARATION OF COLUMNS FOR AFFINITY CHROMATOGRAPHY

Sepharose MH1 columns were prepared as follows:

1g of freeze dried cyanogen bromide activated Sepharose 4B (Pharmacia)was weighed out and suspended in 1 mM HCl. The swollen gel was washedfor 15 minutes in 1 mM HCl on a sintered glass filter.

MH1 antibody (20 mg) was dissolved in coupling buffer (0.1M NaHCO₃, pH8.3 containing 0.5M NaCl) and gently mixed with the swollen gel in astoppered vessel for 2 hours at room temperature or overnight at 4° C.

After mixing, the gel was washed with coupling buffer to remove excessantibody. Unblocked active sites were blocked by treating with 0.1M TrisHCl, pH 8.0 (or 1M ethanolamine, pH 9.0) for 2 hours at roomtemperature. The antibody-bound gel was then washed 3× with 0.1M Acetatebuffer (pH 4.0 containing 0.5M NaCl) followed by Tris buffer (0.1M, pH8.0 containing NaCl 0.5M). Coupled antibody was stored at 4° C. insodium azide solution (0.05%).

Sepharose DesAABB fibrin monomer columns were prepared as follows:DesAABB fibrin monomers were coupled to cyanogen bromide-activatedSepharose 4B in the presence of 1M NaBr in 0.1M borate buffer (pH 8.2)using the procedure described above for preparation of Sepharose MH1.

6.3.6 CHARACTERIZATION OF THE SOLUBLE FIBRIN ENTITY RECOGNIZED BY THE INVITRO ASSAY FOR SOLUBLE FIBRIN POLYMERS USING MH1 TO DETECT SOLUBLEFIBRIN

It has been demonstrated above, Section 5.5, that the MH1 antibody doesnot recognize fibrinogen, the plasma precursor of the fibrin polymer. Itwas also shown above, Section 5.5, that the antibody recognizes bothcrosslinked and non-crosslinked fibrin polymer. In addition, it wasshown above, Section 5.5, that plasmin degradation of fibrin, (or anyprocess which leads to the destruction of the fibrin polymericstructure) causes a loss of immune recognition of fibrin by suchantibody. Consequently, the MH1 antibody does not recognize any of theknown plasmin degradation products of crosslinked fibrin.

Evidence that the MH1 antibody only recognizes the polymeric structureof DesAABB fibrin and does not recognize the monomeric desAABB fibrinentity was obtained by affinity chromatography. Briefly, 0.5 mg of theMH1 antibody was passaged over a 5 ml Sepharose desAABB fibrin monomercolumn. The column was washed with equilibration buffer (0.1 m Trisbuffered saline (TBS), pH 8.5). No significant binding of the antibodyto the column was observed. As shown in FIG. 1A, the antibody eluted inthe run through of the column. When the column was eluted with 6Mguanidine hydrochloride, a small amount of bound protein was recovered.Protein was detected by monitoring all fractions at 280 nm. Antibody wasdetermined by testing immunoreactivity of the protein by means of asolid phase ELISA using fibrin coated microtiter wells. See section5.4.2 above for description of the ELISA used. The protein bound to thecolumn is a result of the presence of a small amount of contaminatingsoluble DesAABB fibrin polymer coupled to the DesAABB fibrin monomercolumn. This contaminant is present in the starting material used in thepreparation of the DesAABB fibrin monomer column. To demonstrate thatthe Sepharose desAABB fibrin monomer column was capable of binding anantibody, a fibrinogen specific monoclonal antibody (Mab) was passagedover the column in a control experiment. As shown in FIG. 1B, the columnbound the control Mab, the antifibrinogen antibody 45J, and treatmentwith guanidine hydrochloride (6M) was required to elute the 45Jantibody.

Evidence that the MH1 antibody, described above, recognizes bothcrosslinked and non-crosslinked soluble DesAABB fibrin polymers wasobtained in an experiment in which soluble DesAABB fibrin polymers weregenerated by addition of thrombin to citrated plasma samples in thepresence and absence of EDTA (EDTA prevents factor XIIIa fromcrosslinking DesAABB fibrin polymers). The crosslinked andnon-crosslinked soluble DesAABB fibrin polymers were prepared asdescribed in section 6.3.1 and their respective immunoreactivities weremeasured using the ELISA as described in section 6.3.4. As shown in FIG.2, there is no significant difference in the immunoreactivity ofcrosslinked (B) and non-crosslinked (C) fibrin polymers in the assaysystem.

Evidence that the MH1 antibody does not recognize soluble desAA fibrinpolymer was obtained in an experiment wherein DesAA fibrin polymer wasprepared by treating fibrinogen or plasma with the snake venom derivedenzyme Batroxobin, from Bothrops atrox, which selectively cleavesfibrinopeptide A (FPA) from the fibrinogen molecule. Batroxobin does notcleave fibrinopeptide B (FPB), in contrast to thrombin which cleavesboth FPA and FPB. The removal of FPA results in polymerization of thedesAA fibrin monomer units and the formation of a DesAA fibrin clot.FIG. 3 demonstrates clot formation, as measured by absorbance of thereaction mixture at 340 nm, when a batroxobin solution is incubated for15 minutes at room treatment with a fibrinogen sample. The absorbanceincreases gradually with time as the soluble DesAA fibrin polymersincrease in length and concentration, until finally the clot is formed.In the experiment to test MH1 assay recognition of desAA fibrin, solubledesAA fibrin polymer was generated in vitro by incubation of afibrinogen sample with batroxobin (final concentration, 0.5 units/mL) at37° C. An aliquot was removed from the batroxobin treated sample after 7minutes and tested in the ELISA assay of section 6.3.4. No reactivitywas demonstrated with this sample, sample C, as shown in FIG. 4. It canalso be deduced from this experiment that the assay system does notrecognize fibrinogen-desAA fibrin complex which is another source ofsoluble fibrin. Clearly the reaction mixture produced in this experimentwould contain such "soluble fibrin" entities since the batroxobinproduces desAA fibrin monomers which are free to interact with otherdesAA monomers or non-digested fibrinogen molecules. Since no reactionwas demonstrated in the assay system after addition of batroxobin, theconclusion can be drawn that the assay does not detect thesefibrinogen-desAA fibrin entities. Sample B, which is the positivecontrol, was composed of DesAABB fibrin polymers formed by treatment ofa plasma sample with thrombin and hirudin. Untreated fibrinogen, sampleA, was added as a negative control.

Soluble fibrin complex can also arise due to factor XIIIa crosslinkingof native fibrinogen molecules to form fibrinogen dimers (Kanaide etal., J. Lab. Clin. Med. 86:574-579 (1975)) Factor XIIIa (FXIIIa) treatedfibrinogen is not detected by the assay system. In this experiment,fibrinogen coated microtiter plates were first treated withthrombin-activated factor XIIIa for 11/2 hours at 37° C. After quenchingresidual thrombin activity, the MH1 antibody was incubated in the FXIIIatreated wells and bound MH1 was detected by use of anti-mouse alkalinephosphatase conjugate. Bound anti-mouse antibody was detected by theaddition of an alkaline phosphatase substrate. The level of MHI bound tothe wells was measured by reading the optical density at 490 nm. FIG. 5,sample labeled Fg+FXIIa shows that no MH1 was bound to the wells,indicating that the antibody does not recognize crosslinked fibrinogenstructures. As a positive control the fibrinogen coated wells weretreated with thrombin to produce desAABB fibrin polymers and tested forimmunoreactivity with MH1 antibody, FIG. 5, sample labeled Fg+Thrombin.

6.3.7 MEASUREMENT OF SOLUBLE DESAABB FIBRIN POLYMER FORMATION IN VITRO

Soluble DesAABB fibrin polymers were produced in vitro by addition oflow levels of thrombin (0.025 NIH units/mL) to a citrated plasma sample.The sample was incubated at 37° C. Aliquots were removed from the sampleat 1 minute intervals and the thrombin activity was quenched by theaddition of the potent thrombin inhibitor, hirudin (final concentration2-ATU/ml). The amount of soluble DesAABB fibrin polymer in each aliquotwas measured by the ELISA procedure described in section 6.3.4.

As shown in FIG. 6, the amount of soluble fibrin polymer, indicated bythe absorbance at 490 nm, rises sharply after an initial lag period of4-5 minutes after the addition of thrombin to the plasma. The level ofsoluble fibrin polymers peaks at approximately 7.5 minutes after theaddition of thrombin to the plasma. The steep decrease in the solublefibrin level observed after 9-10 minutes coincides with gelation andformation of insoluble fibrin polymers (clot formation).

6.3.8 PURIFICATION OF SOLUBLE FIBRIN POLYMERS FROM PLASMA BYSEPHAROSE-MH1 AFFINITY CHROMATOGRAPHY

A plasma sample was incubated with thrombin (0.025 units ml) for 7minutes at 37° C. The thrombin activity was quenched by the addition ofexcess hirudin. The reaction mixture was passaged over a Sepharose-MH1column. The starting material, the run through (nonbound material) thebound protein (purified protein) which protein was eluted using NaSCN(3) and dialyzed against PBS, were tested for immunoreactivity with MH1using the ELISA assay of section 6.3.4. The immunoreactivity of thethrombin treated plasma after quenching with hirudin (StartingMaterial), of the bound protein after removal from the column anddialysis (Elution), and of the nonbound material (Nonbound) is shown inFIG. 7. The starting material and the bound protein after dialysis wereimmunoreactive with MH1 while the nonbound material was not.

6.3.9 IN VITRO DETECTION AND MEASUREMENT OF SOLUBLE CROSSLINKED ANDSOLUBLE NON-CROSSLINKED FIBRIN POLYMERS FOR DIAGNOSIS OF MI

36 different plasma samples were tested for soluble crosslinked andsoluble non-crosslinked DesAABB fibrin polymer levels using the ELISAassay described above in section 6.3.4 with MH1 as the capture antibody.After collection, plasma samples were drawn into anticoagulant andfrozen. The thawed samples were tested for soluble crosslinked andnon-crosslinked DesAABB fibrin polymer. Ten of these 36 samples weretaken from healthy control humans. Healthy controls were recruited fromlaboratory personnel who were in excellent health and who did notexperience chest pain even after exertion. All other samples were frompatients at the emergency room facility of a local community hospital.All emergency room patients had clinical symptoms (chest pain)suggestive of myocardial infarction (MI); 15 were confirmed as sufferingfrom MI; the other 11 patients were all confirmed not to be sufferingfrom MI. The latter patient group was diagnosed with various clinicalconditions including, angina pectoris, anxiety, pulmonary edema. Theblood samples were drawn from patients immediately after their admissionto the emergency room. In most cases information on the duration of thesymptoms were recorded. In this study all patients had been experiencingchest pain for 1-3 hours prior to admission. As shown in FIG. 8, in allcases the patients with confirmed MI had significantly higher levels ofsoluble DesAABB fibrin polymers than the healthy controls (3-30 folddifference). In those patients with chest pain but without MI, thelevels of soluble DesAABB fibrin polymers were not significantlydifferent from those of the healthy controls. (Levels of solublecrosslinked and soluble non-crosslinked DesAABB fibrin polymers areshown as "Level of Soluble Fibrin Elisa Units (×1000)").

The sensitivity of this assay system was therefore 100% (for n=36) andthe specificity was also 100% (for n=36) i.e., there were no falsepositives and no false negatives. It can therefore be concluded thatthis assay system has a high level of diagnostic accuracy for myocardialinfarction.

6.4 KITS FOR IN VITRO DETECTION OF SOLUBLE CROSSLINKED FIBRIN POLYMERSAND SOLUBLE NON-CROSSLINKED FIBRIN POLYMERS

It is to be understood that the present invention is not limited to theuse of monoclonal antibodies in the assay. However, where such antibodyis used, with respect to the kit hereinafter, these kits contain a setof standards, a first antibody (i.e., capture antibody, for example MH1)which can be immobilized on a surface and a second antibody labeled witha signal generator as described above. These kits contain standards inthe form of known amounts of soluble cross-linked DesAABB fibrin polymerand soluble non-crosslinked DesAABB fibrin polymer. Such standards maybe prepared by the isolation of soluble crosslinked and solublenon-crosslinked DesAABB fibrin polymers using the MH1 Sepharose affinitycolumn as described above. The kits may also contain specific buffers,separating agents and controls. The kits may contain collection devicesor chemicals to treat the sample to be assayed.

All publications and patents cited above are herein incorporated byreference.

We claim:
 1. A fibrin-specific monoclonal antibody that is MH-1 producedby hybridoma ATCC 9739 or an immunoreactive fragment thereof, or anantibody or an immunoreactive fragment thereof that competitivelyinhibits the immunospecific binding of the MH-1 antibody produced byhybridoma ATCC HB 9739 to MH-1's target antigen, wherein saidfibrin-specific monoclonal antibody or an immunoreactive fragmentthereof is conjugated to a cytotoxic reagent.
 2. The conjugatedfibrin-specific monoclonal antibody of claim 1, wherein the cytotoxicreagent is covalently linked to the fibrin-specific monoclonal antibody.3. The conjugated fibrin-specific monoclonal antibody of claim 1,wherein the fibrin-specific monoclonal antibody is MH-1.
 4. Theconjugated fibrin-specific monoclonal antibody of claim 1, wherein thecytotoxic reagent is a protease capable of lysing fibrin.
 5. Theconjugated fibrin-specific monoclonal antibody of claim 4, wherein theprotease is streptokinase.
 6. The conjugated fibrin-specific monoclonalantibody of claim 4, wherein the protease is tissue plasminogenactivator.
 7. The conjugated fibrin-specific monoclonal antibody ofclaim 4, wherein the protease is urokinase.